Compositions for transfecting a nucleic acid molecule into a cell comprising triazole compounds grafted to a cationic polymer, and their applications

ABSTRACT

Disclosed are compositions for transfecting a nucleic acid molecule into a cell and their applications. Specifically, this relates to a composition suitable for transfecting a nucleic acid molecule into a cell, preferably a eukaryotic cell, including (i) at least one compound of general formula (I), preferably of general formula (III), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or an acceptable salt thereof, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium, wherein Y 1 , Y 2 , Y 3 , Z 1 , Z 2 , Z 3 , X 1 , X 2 , R 3 , P + , R and V are as defined in the description. Also disclosed are uses of the composition and to a method for in vitro or ex vivo transfection of live cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase of International ApplicationNo. PCT/EP2020/072068 filed Aug. 5, 2020 which designated the U.S. andclaims priority to EP Patent Application No. 19315083.6 filed Aug. 5,2019, the entire contents of each of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to compositions for transfecting a nucleicacid molecule into a cell comprising heterocyclic compounds, inparticular triazole derivatives grafted to a cationic polymer, and theirapplications. The present invention is directed to a compositionsuitable for transfecting a nucleic acid molecule into a cell,preferably a eukaryotic cell, comprising (i) at least one compound ofgeneral formula (I), preferably at least one compound of general formula(III), or a tautomer, mesomer, racemate, enantiomer, diastereomer, ormixture thereof, or an acceptable salt thereof, and (ii) an acceptableexcipient, buffering agent, cell culture medium, or transfection medium,wherein Y¹, Y², Y³, Z¹, Z², Z³, X₁, X₂, R₃, P⁺, R and V are as definedin the description. The present invention also relates to uses of saidcomposition and to a method for in vitro or ex vivo transfection of livecells.

Description of the Related Art

The gene transfer is the process of introducing copies of exogenousgenes into living cells in order to induce synthesis of the gene'sproducts. Transfection is the process of deliberately and artificiallyintroducing nucleic acids (DNA or RNA) into eukaryotic cells, utilizingmeans of non-viral methods. The transfection is of fundamentalimportance to developments in modern biology and medicine, and hasprovided much of our knowledge of gene function and regulation.

The transfection according to the invention can be achieved in variouscells, including mammalian and insect cells, in primary cells, celllines, stable cells or tumoral cells. The transfection is a powerfultool for in vitro genomic studies by offering the possibility to expressin cells new exogenous proteins or to over-express or silence naturallyoccurring proteins.

Transfection according to the invention can be applied in therapythrough ex vivo or in vivo protocols. Nucleic acid-based therapy withnon-viral vectors can target different diseases, genetic diseases,immune diseases, cancers or viral infections in various tissues/organsor tumors. The cell targeting is achieved through different mechanismsand depends on the nature and properties of the transfection reagent,method or protocol composition or formulation and the route ofadministration (Kaestner et al., 2015).

In bioproduction, transfection according to the invention can be used togenerate stable cell clones over-producing recombinant proteins,peptides or antibodies. More recently, the transfection allowingtransient gene expression (TGE) is becoming a valuable method for thefast production of moderate level of recombinant proteins or antibodiesuseful for research and process development phases. Transient geneexpression processes are advantageously applied for the production ofrecombinant viruses such as adeno-associated viruses (AAV), lentiviruses(LV) or adenoviruses (Merten et al., 2016; Van Der Loo and Wright,2015). Such processes consist of transfecting many expression vectors(plasmids) expressing in cells the different components necessary toproduce the viruses including capsid proteins, helper proteins, envelopproteins, viral polymerase or regulators, or viral genomes. Highproducing cells are used in viral production such as HEK293 andderivative cells, HeLa, BHK-21, A549 or insect cells. The transfectioncan be achieved in adherent or suspension-adapted cells at high celldensity cultured in media containing serum or in protein-free,chemically defined or completely synthetic media.

Transfection is a method to introduce the different components in cellsnecessary to induce genome modification, engineering or editing such aszing finger nucleases, CRE/LOX proteins or CRISPR Cas-9 proteins.

DNA transfection uses plasmid DNA which triggers the gene expressiondriven by a promoter of a protein or peptide and/or a nucleic acid suchas messenger RNA, long RNA, microRNA, short hairpin RNA, shortinterfering RNA, . . . .

In mainly all cases plasmid DNA has been used for transfection purposesbecause of its inherent stability and its ability to integrate into thehost genome to produce stable gene expression or to remain in thenucleus under an episomal form providing transient gene expression.However, some cells, named ‘hard to transfect’ cells (HTT) arerefractory to DNA transfection or exhibit low levels of transfection andgene expression when compared to standard transformed cells linesroutinely used in laboratory settings. These “hard to transfect” cellsexhibit less than 50% transfection efficiency when transfected with thelast generation of commercially available transfection reagents such asLipoFectAmine® 2000 & 3000 (ThermoFisher), TransIT Reagents® (MirusBio),FuGene® (Promega), XtremeGene® (Roche), jetPRIME®(Polyplus-transfection) or ViaFect® (Promega).

Recent progresses to improve the gene expression efficiency of HTT cellsare the transfection with messenger RNA (mRNA) sequences rather thanplasmid DNA constructs which showed significant increase of transfectionand gene expression levels in a majority of cell types, and particularlyin challenging HTT cells. The benefice is explained by the fact that thetransfected mRNA does not need to reach the nucleus for cellular actioncontrasting with DNA transfection where the major limitation is to reachand penetrate the nucleus. The plasmid DNA import is not well understoodbut an efficient DNA transfection is mainly correlated with an activeproliferation rate of cells where the transfected DNA may diffuse in thenuclear space during the nuclear membrane breakdown. In mostpost-mitotic cells or non-dividing cells, DNA transfection is noteffective. Most of the HTT cells exhibits a low level or absence ofmitosis such as neuronal cells or other cell types derived from neuraltissue, primary blood cells like dendritic cells or macrophages, orprimary hepatocytes. However, for other HTT cells, the low transfectionefficiency might be explained by other factors such as the cellfragility, the low binding of transfection material to the cell plasmamembrane, the low endocytosis capacity or a non-efficient intracellulartrafficking towards the nucleus of the transfected DNA.

Transfection of plasmid DNA is the most common method to overexpressproteins in cells grown in culture. Most of the methods to introducegenetic DNA material into cells include the use of reagents such ascalcium phosphate, cationic liposomes, peptides or polymers. When thetransfection fails, the reagent is generally recognized as the culprit.There is still a need to improve the efficiency of transfection reagentsparticularly for the HTT cells, with new concepts and generation ofreagents.

DNA transfection in eukaryotic cells involves combining or mixing thepolyanionic DNA molecule with a reagent to form transfection complexesor aggregates. Among the most commonly used reagents, cationic lipids,peptides or polymers are suitable to interact with the negativelycharged DNA. If an excess of the cationic reagents is used, complexes oraggregates having a positive character are generated. Such complexes areable to interact with the negatively charged glycosaminoglycans such asheparan sulfates present on the cell plasma membranes (Labatmoleur etal., 1996, Mislick and Baldeschwieler, 1996). The cell membrane bindingof complexes induces a cell internalization or uptake by endocytosismechanism. Transfection complexes are transported into endosomes wheretransfection reagents exhibit membrane destabilization though fusogenicactivity and/or endosomolysis to release DNA in the cytoplasm. Followingthe release from the endosomes, the transfected DNA has to diffusetowards the perinuclear space and penetrate in the nucleus. The nuclearimport is a limiting step as plasmid DNA is not able to diffuse throughthe nuclear pore complexes because of its large size.

Among the non-viral vectors for DNA transfection, cationic liposomes oraggregates are one of the major classes which consist of combining orformulating cationic lipids with other types of lipids, such asphospholipids or cholesterol, to generate positively charged liposomes,vesicles or micelles that can bind negatively charged DNA and bindnegatively charged cell membranes ending by cell transfection. In theprior art, the first synthetic cationic lipid isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)by Felgner et al. When combined with dioleoylphosphatidylethanolamine ata ratio of 1:1, DOTMA formed cationic liposomes that were able totransfect cells in vitro. Based on the positively chargedtrimethylammonium polar head other monocationic lipids were developedsuch as 1,2bis(oleoyloxy)-3,3-(trimethylammonium) propane chloride(DOTAP). Other prior art compounds are based on polycationic polar headsuch as lipids described by Behr et al., 1989,dioctadecylamidoglycylspermine (DOGS) or dipalmitoylphosphatidylethanolamidospermine (DPPES) where the carboxyspermine wasused instead of ammonium group or the phospholipid moiety was replacedby a cholesterol derivative (Gao & Huang, 1991) such as3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride(DC-CHOL). Since these pioneer works, many cationic lipids were designedwith the goal to generate novel cationic lipid reagents with increasedtransfection efficiencies. Many of these reagents are commerciallyavailable and the recent LipoFectAmine3000® reagent represents the mostadvanced reagent of cationic lipids available on the market. However,limitations are still observed as transfection is not effective in allcell types and cell toxicity is still a major concern of cationic lipidsystems.

Cationic polymers represent the second major class of transfectionreagents with the advantage to offer a large density of charged aminogroups in their backbones. Cationic polymers having a positive charge atphysiological pH are able to complex DNA into particles or aggregates,initiate cell binding and trigger cell internalisation throughendocytosis. Polylysine (PLL) was the first polymer used but showed verylimited transfection efficiencies (Wu and Wu, 1987, Zenke et al., 1990).Its efficiency can be improved when additives such as weak bases likechloroquine (Erbacher et al., 1996) or fusogenic peptide like influenzapeptides (Planck et al., 1994) were added in order to buffer the acidicpH of destabilize endosomes, respectively, and induce more release ofDNA in the cytoplasm. Behr et al. has showed that the polyethylenimine(PEI) was a more effective polymer than PLL in transfection (Boussif etal., 1995). PEI has a high density of amino groups and is not fullyprotonated at physiological pH. After endocytosis of DNA complexed withPEI, the polymer has buffering capacity which induces a ‘proton sponge’activity resulting in vesicles swelling and endosomolysis ending by therelease of DNA in the cytoplasm without the help of additives (Boussifet al., 1995; Sonawane et al., 2003). Both branched and linear PEI areefficient in transfection but the linear topology was shown to be moreefficient (Itaka et al., 2004), not inhibited by the presence of serumand less toxic when compared to the branched form. Since two decades,many strategies were developed to increase the transfection efficiencyof PEI, reduce its toxicity or propose alternative of biodegradablePEI-based polymers.

Many works were concentrated on the optimisation of the intrinsicproton-sponge endosomolytic activities of PEI by grafting histidyl orbenzyl residues (U.S. Pat. No. 8,658,150, Chandrashekhar et al., 2012)to the polymers. Other modifications were explored like addition ofhydrophilic groups (EP2070970) to increase the solubility of DNA/PEIcomplexes and reduce the cell toxicity. Hydrophobic functionalities wereadded to PEI to increase the biodegradability of the polymer usingN-acyl groups (EP0262641) or to generate lipopolymers (US20090022746,WO2006/041617). Higher gene transfection efficiencies were observed invarious cell lines. However, the efficiency in “hard to transfect” cellsremained very limited.

Other cationic polymers were described for DNA transfection such aschitosan (Erbacher et al., 1998), polyamidoamine (PAMAM) dendrimers(Tomalia et al., 1985, Haensler and Szoka, 2003), degraded or fractureddendrimers (Tang et al., 1996), structurally flexible dendrimers (Liu etal., 2011), polyaminoesters (Little et al., 2004),poly(a[4-aminobutyl]-L-glycolic acid) (Akinc et al., 2003), cationiccyclodextrin amphiphiles (Cryan et al., 2004), poly(N-methylvinylamine)(Dréan et al., 2018), poly(2-N-dimethylaminoethyl)methacrylate(PDMAEMA), polyallylamine (Boussif et al., 1999), polyornithine (Dong etal., 1993), polyarginine (Alhakamy et al., 2013), polyhistidine (Putmanet al., 2003) and cell penetrating peptides (CPPs) (Gupta, 2005).

It was reported that cationic polymers such as PEI were able totransfect post-mitotic cells (Brunner et al.). However, in the absenceof mitosis and the subsequent breakdown of the nuclear membrane, it wasshown that plasmid DNA, because of its large size >1 kbp, was not ableto enter the nucleus through the nuclear pore complexes (Lukacs et al.2000). Once released from endosomes, DNA was still associated with somecationic polymers which contributed to protect it against the nucleasedegradation (Lechardeur et al., 1999). It is known that DNA is able tointeract with proteins present in the cytoplasm, particularly dynein,allowing a microtubule-based movement towards the nucleus or binding oftranscription factors having NLS signals, which may direct DNA to thenuclear pore complexes through the importin pathway (Bai et al., 2017).

Cationic polymers represent one class of delivery reagents suitable forin vivo applications for gene therapy approach where DNA/cationicpolymer complexes are directly injected through different routes ofadministration, such as intravenous, intraperitoneal, intradermal,intratumoral or intracerebral injection. Cationic polymers formulatedwith an acceptable excipient and/or buffering agent are suitable for invivo gene transfer. Particularly, PEI was reported as an efficientpolymer for in vivo applications (Boussif et al., 1995).

Due to their special structural features and electron-rich environment,heterocyclic compounds such as pyrazole, imidazole or triazolederivatives, in particular triazole derivatives exhibit a broad spectrumof bioactivities. Triazoles derivatives may have properties to influencethe pH in endosomes. In addition, the triazole may contribute tohydrogen bonds with nucleic acids. The addition of cycloalkyl or arylmoieties to triazole may offer supplementary hydrophobic interactionssuch as π-π stacking with nucleobases. All together, these propertiesmay fine-tune the interactions with nucleic acids and offer thepossibilities to develop new DNA carriers.

SUMMARY OF THE INVENTION

The inventors provide away to improve transfection reagent by usingaromatic heterocyclic compounds, in particular triazole derivatives tofine-tune the affinity and binding to a nucleic acid molecule, e.g. DNA,optimize the buffering capacity in acidic conditions and/or increase thediffusion, binding and uptake in the nucleus.

Thus it is an object of the present invention to provide a moreefficient transfection composition or a formulation for transfecting anucleic acid molecule into a cell.

It is another object of the present invention to provide a method fortransfecting a nucleic acid molecule using said composition orformulation comprising such composition for administration to cells.

The inventors carried out a structure-based screening of substitutedheterocyclic compounds, in particular imidazole, triazole, pyrazolederivatives, to improve the efficiency of transfection by cationicpolymers. Such substituted heterocyclic compounds were grafted tocationic polymers, in particular polyethylenimine (PEI) polymers, ofvarious molecular weight in order to fine-tune the conjugates. Manyvariations were proposed in order to define optimal structuresfacilitating transfection of a nucleic acid molecule, e.g. DNA.Heterocycles exhibiting hydrophobic properties were developed and mayrepresent binding motifs to cytoplasmic proteins promoting potentiallythe nuclear import.

The present invention relates to a composition suitable for transfectinga nucleic acid molecule into a cell, preferably a eukaryotic cell,comprising (i) at least one compound of general formula (I) or atautomer, mesomer, racemate, enantiomer, diastereomer, or mixturethereof, or an acceptable salt thereof, and (ii) an acceptableexcipient, buffering agent, cell culture medium, or transfection medium:

wherein:

-   -   Y¹, Y² and Y³, which may be identical or different, represent C        or N, with the proviso that at least two of Y¹, Y² and Y³ are N,        and with the further proviso that at least one, but no more than        two, of Y¹, Y² and Y³ are substituted by Z¹, Z² and Z³        respectively;    -   Z¹ represents H, X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺,        X₁—P⁺, R₃—P⁺, or X₂—P⁺; or    -   Z¹ is absent;    -   Z² represents H, a linear or branched, saturated or unsaturated        C₁-C₁₈ alkyl, C₆-C₁₈ aryl, a linear or branched, saturated or        unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, a linear or branched,        saturated or unsaturated C₂-C₁₈ heteroalkyl, C₅-C₁₀ heteroaryl,        halogen, OH, a linear or branched, saturated or unsaturated        C₁-C₁₈ alkylamine, a C₁-C₁₂ alkoxy, a linear or branched,        saturated or unsaturated C₁-C₁₈ alkyl-C₁-C₁₂ alkoxy,        X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or        X₂—P⁺; or Z² is absent;    -   Z³ represents H, a linear or branched, saturated or unsaturated        C₁-C₁₈ alkyl, C₆-C₁₈ aryl, a linear or branched, saturated or        unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, C₅-C₁₀ heteroaryl, a        linear or branched, saturated or unsaturated C₂-C₁₈ heteroalkyl,        C₂-C₁₈ alkylidene, OH, guanidine, halogen, X₁—R₃—X₂—P⁺,        X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺; or Z³ is        absent;    -   X₁ and X₂, which may be identical or different, represent CO or        CH₂;    -   R₃ represents (CH₂)_(m), (CH₂)_(m)—CHCH₃—(CH₂)_(n)—,        (CH₂)_(m)—C(CH₃)₂—(CH₂)_(n)—, (CH₂)_(m)—O—(CH₂)_(n)—,        (CH₂)_(m)—S—(CH₂)_(n)—, (CH₂)_(m)—CH₂—O—, with m representing an        integer between 1 and 3, preferably m is equal to 2 and n        representing an integer between 1 and 3;    -   P⁺ represents a graft cationic polymer, which is a polyamine        comprising secondary amines, tertiary amines, a mixture of        primary and secondary amines, a mixture of primary and tertiary        amines, a mixture of secondary and tertiary amines, or a mixture        of primary, secondary and tertiary amines;    -   R or V represents H, a linear or branched, saturated or        unsaturated C₁-C₁₈ alkyl or cycloalkyl, a C₆-C₁₈ aryl, a linear        or branched, saturated or unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl,        a linear or branched, saturated or unsaturated C₂-C₁₈        heteroalkyl, a linear or branched, saturated or unsaturated        C₁-C₂₄ ester, a C₅-C₁₀ heteroaryl, a C₅-C₁₀ heterocyclyl, a        linear or branched, saturated or unsaturated C₁-C₁₈ alkyl-C₅-C₁₀        heteroaryl, X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺,        R₃—P⁺, or X₂—P⁺; with the proviso that:    -   only one of Z¹, Z², Z³, R or V represents X₁—R₃—X₂—P⁺, X₁—R₃—P⁺,        X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺, preferably        X₁—R₃—X₂—P⁺.

In a preferred embodiment of the invention, the composition suitable fortransfecting a nucleic acid molecule into a cell, preferably aeukaryotic cell, comprises (i) at least one compound of general formula(III) or a tautomer, mesomer, racemate, enantiomer, diastereomer, ormixture thereof, or an acceptable salt thereof, and (ii) an acceptableexcipient, buffering agent, cell culture medium, or transfection medium:

wherein:

-   -   Z¹ represents H, X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺,        X₁—P⁺, R₃—P⁺, or X₂—P⁺; or    -   Z¹ is absent;    -   Z² represents H, a linear or branched, saturated or unsaturated        C₁-C₁₈ alkyl, C₆-C₁₈ aryl, a linear or branched, saturated or        unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, a linear or branched,        saturated or unsaturated C₂-C₁₈ heteroalkyl, C₅-C₁₀ heteroaryl,        halogen, OH, a linear or branched, saturated or unsaturated        C₁-C₁₈ alkylamine, a C₁-C₁₂ alkoxy, a linear or branched,        saturated or unsaturated C₁-C₁₈ alkyl-C₁-C₁₂ alkoxy,        X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or        X₂—P⁺; or Z² is absent;    -   Z³ represents H, a linear or branched, saturated or unsaturated        C₁-C₁₈ alkyl, C₆-C₁₈ aryl, a linear or branched, saturated or        unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, C₅-C₁₀ heteroaryl, a        linear or branched, saturated or unsaturated C₂-C₁₈ heteroalkyl,        C₂-C₁₈ alkylidene, OH, guanidine, halogen, X₁—R₃—X₂—P⁺,        X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺; or Z³ is        absent;    -   X₁ and X₂, which may be identical or different, represent CO or        CH₂;    -   R₃ represents (CH₂)_(m), (CH₂)_(m)—CHCH₃—(CH₂)_(n)—,        (CH₂)_(m)—C(CH₃)₂—(CH₂)_(n)—, (CH₂)_(m)—O—(CH₂)_(n)—,        (CH₂)_(m)—S—(CH₂)_(n)—, (CH₂)_(m)—CH₂—O—, with m representing an        integer between 1 and 3, preferably m is equal to 2 and n        representing an integer between 1 and 3;    -   P⁺ represents a graft cationic polymer, which is a polyamine        comprising secondary amines, tertiary amines, a mixture of        primary and secondary amines, a mixture of primary and tertiary        amines, a mixture of secondary and tertiary amines, or a mixture        of primary, secondary and tertiary amines;    -   R or V represents H, a linear or branched, saturated or        unsaturated C₁-C₁₈ alkyl or cycloalkyl, a C₆-C₁₈ aryl, a linear        or branched, saturated or unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl,        a linear or branched, saturated or unsaturated C₂-C₁₈        heteroalkyl, a linear or branched, saturated or unsaturated        C₁-C₂₄ ester, a C₅-C₁₀ heterocyclyl, a C₅-C₁₀ heteroaryl, a        linear or branched, saturated or unsaturated C₁-C₁₈ alkyl-C₅-C₁₀        heteroaryl, X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺,        R₃—P⁺, or X₂—P⁺; with the provisos that:    -   at least one of Z¹, Z² or Z³ is present, preferably Z¹ or Z³ is        present; and    -   only one of Z¹, Z², Z³, R or V represents X₁—R₃—X₂—P⁺, X₁—R₃—P⁺,        X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺.

In a particular embodiment of the above composition, only one of Z¹, Z²or Z³ is present, preferably Z¹ or Z³ is present.

As defined herein, the term “tautomer” refers to structural isomersdiffering only in the positions of hydrogen atoms and electrons.Examples of tautomers include, but are not limited to, ketone-enol,enamine-imine, amide-imidic acid, lactam-lactim, nitroso-oxime,ketene-ynol, amino acid, or phosphite-phosphonate.

As defined herein, the term “mesomer” or “meso compound” refers to astereoisomer that has two or more chiral centers but is opticallyinactive.

As defined herein, the term “racemate” or “racemic mixtures” refers to amixture of two enantiomers in equal proportions.

As defined herein, the term “enantiomer” refers stereoisomers that aremirror images, i.e. mirror image isomers.

As defined herein, the term “diastereomer” refers to isomers ofcompounds with more than one chiral center that are not mirror images ofone another.

As defined herein, the term “acceptable excipient” refers to apharmaceutically acceptable vehicle, which is any substance orcombination of substances physiologically acceptable i.e., appropriatefor its use in a composition in contact with a host, especially a human,and thus non-toxic. It can refer to a solid, semi-solid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anyconventional type. Examples of suitable acceptable excipients include,but are not limited to, glucose, galactose, lactose, dextrose, maltose,mannitol, sucrose, trehalose, polyethyleneglycol, or pluronic acid.

As defined herein, the term “buffering agent” refers to an agent thatadjusts, maintains or controls the pH of a solution. Buffering agentscan be either the weak acid or weak base that would comprise a buffersolution. Examples of suitable buffering agents include, but are notlimited to, sodium carbonate, sodium bicarbonate, sodium hydroxide,calcium bicarbonate, calcium citrate, sodium citrate, magnesiumhydroxide, magnesium bicarbonate, potassium acetate, Tris acetate,sodium acetate, potassium phosphate monobasic, potassium carbonate,potassium bicarbonate, potassium citrate, or magnesium oxide.

As defined herein, the term “cell culture medium” or “transfectionmedium” refers to a medium containing serum, synthetic medium,animal-free component medium or chemically defined medium, in particularmedium for maintaining cells alive, or for growing, for differentiatingor for expanding cells, or for enhancing transfection.

As defined herein, the term “C₁-C₁₈ alkyl” represents any monovalentradical of a linear or branched hydrocarbon chain comprising 1 to 18carbon atoms. The term “C₁-C₆ alkyl” represents an alkyl group having 1to 6 carbon atoms. Examples of suitable C₁-C₁₈ alkyl groups include, butare not limited to, C₁-C₄ alkyl groups such as methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl, s-butyl or t-butyl, C₆-C₈ alkyl groups suchas n-hexyl, n-heptyl or n-octyl, as well as n-pentyl, 2-ethylhexyl,3,5,5-trimethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl orn-octadecyl.

As defined herein, the term “C₁-C₁₂ alkoxy” represents a radical offormula —OR′, wherein R′ is a C₁-C₁₂ alkyl. Examples of suitable C₁-C₁₂alkoxy groups include, but are not limited to, C₁-C₆ alkoxy groups suchas methoxy (—OCH₃), ethoxy (—OCH₂CH₃), t-butoxy (—OC(CH₃)₃), or—O(CH₂)₅CH₃.

As defined herein, the term “C₆-C₁₈ aryl” represents any monovalentradical of an aromatic hydrocarbon comprising 6 to 18 carbon atoms.Examples of suitable C₆-C₁₈ aryl groups include, but are not limited to,phenyl, naphthyl, anthracenyl or phenanthrenyl.

As defined herein, the term “C₆-C₁₈ aryl-C₁-C₁₈ alkyl” represents anaryl group as defined herein combined to an alkyl group as definedherein. Examples of suitable C₆-C₁₈ aryl-C₁-C₁₈ alkyl groups include,but are not limited to, benzyl, phenylethyl (or phenethyl),phenylpropyl, phenylbutyl, phenylpentyl, phenylhexyl, naphthylmethyl,naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylpentyl,naphthylhexyl, anthracenylmethyl, anthracenylethyl, anthracenylpropyl,anthracenylbutyl, anthracenylpentyl, anthracenylhexyl,phenanthrenylmethyl, phenanthrenylethyl, phenanthrenylpropyl,phenanthrenylbutyl, phenanthrenylpentyl or phenanthrenylhexyl.

As defined herein, the term “C₂-C₁₈ heteroalkyl” represents an alkylgroup as defined herein substituted by one or more heteroatoms such asO, N, or S.

As defined herein, the term “C₅-C₁₀ heteroaryl” represents anymonovalent radical of a monocyclic or bicyclic 5 to 10 membered aromaticgroup comprising from 1 to 3 heteroatoms independently selected fromoxygen, nitrogen and sulfur. Examples of suitable C₅-C₁₀ heteroarylgroups include, but are not limited to, furyl, thienyl, pyrrolyl,pyrazoyl, imidazolyl, isoxazolyl, isothiazoyl, thiazolyl, oxazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1-benzofuryl, 1-benzothienyl, indolyl,benzimidazolyl, indazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl,1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzothiazolyl,benzoxazolyl, benzotriazolyl, pyridyl, pyridinium, quinolinyl,quinolinium, isoquinolinyl, isoquinolinium, pyridazinyl, cinnolinyl,phthalazinyl, pyrimidinyl, quinazolinyl, pyrazinyl or quinoxalinyl.

As defined herein, the term “C₁-C₁₈ alkylamine” represents anymonovalent radical of a linear or branched hydrocarbon chain comprising1 to 18 carbon atoms, in which one of the hydrogen atom bonded to acarbon atom is replaced by an amino group. Examples of suitable C₁-C₁₈alkylamine include, but are not limited to, —(CH₂)_(n)—NH₂, with nrepresenting an integer between 1 and 18, —CH₂NHCH₃, —CH₂CH(CH₃)—NH₂, or—(CH₂)_(n) N(CH₃)₂, with n representing an integer between 1 and 6.

As defined herein, the term “C₁-C₁₈ alkyl-C₁-C₁₂ alkoxy” represents analkyl group as defined herein combined to an alkoxy group as definedherein.

As defined herein, the term “C₂-C₁₈ alkylidene” refers to a divalentgroup derived from an alkane by removal of two hydrogen atoms from thesame carbon atom, the free valencies being part of a double bond (═CR₂).Examples of suitable C₂-C₁₈ alkylidene include, but are not limited to,═CH₂, ═CH(CH₂CH₃), or ═C(CH₃)₂.

As defined herein, the term “halogen” represents an atom of F, Cl, Br orI.

As defined herein, the term “C₁-C₂₄ ester” represents a radical offormula —C(O)OR″, wherein R″ is a C₁-C₂₄ alkyl, in particular a C₁-C₁₈alkyl as defined herein.

As defined herein, the term “C₅-C₁₀ heterocyclyl” refers to anymonovalent radical of a monocyclic or bicyclic 5 to 10 membered ringcontaining one or more heteroatoms such as O, N, or S. Examples ofsuitable heterocyclyl groups include, but are not limited to,piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl,thiomorpholinyl or azepanyl.

Unless mentioned otherwise, the groups and radicals defined hereinabovemay be unsubstituted or substituted by one or more substituents such as,for example, halogen, alkyl, alkoxy, aryl, heteroaryl, haloalkyl,haloalkoxy, alkoxycarbonyl, alkanoyl, aroyl, formyl, nitrile, nitro,amido, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, arylsulfinyl,arylsulfonyl, amino, alkylamino, arylamino, dialkylamino anddiarylamino.

In a particular embodiment of the invention, the composition furthercomprises at least one nucleic acid molecule to be transfected in acell. Preferably said nucleic acid molecule is selected from the groupconsisting of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), aDNA/RNA hybrid, a short interfering RNA (siRNA), a microRNA (miRNA), ashort hairpin RNA (shRNA), a messenger RNA (mRNA), a CRISPR guide RNA,and an expression vector encoding said nucleic acid molecule, inparticular a plasmid encoding said nucleic acid molecule, or a plasmidexpressing said nucleic acid molecule such as siRNA, microRNA, shRNA,CRISPR guide RNA. Preferably said nucleic acid molecule is a DNA.

When distinct nucleic acids are provided in the composition of theinvention, they may be all DNA molecules or all RNA molecules or may bemixtures of DNA and RNA molecules or molecules comprising an associationof DNA and RNA strands.

Said nucleic acid molecule may be single stranded or double stranded,and may contain modified or unmodified bases.

The terms “polynucleotide”, “nucleic acid”, “oligonucleotide”, and“nucleic acid molecule” are used interchangeably herein to designatethese nucleic acid molecules.

The composition according to the invention may be used as a formulationof the nucleic acid molecule with the at least one compound of generalformula (I) (including any of its particular embodiments disclosedherein), preferably the at least one compound of general formula (III),and the acceptable excipient, buffering agent, cell culture medium, ortransfection medium, in accordance with the disclosure provided herein.It may alternatively be used as a cell culture or as expanded cells,wherein prior to being provided as a culture and/or as expanded cells,isolated cells have been treated with said formulation for transfection.Otherwise stated, the composition of the invention encompasses, as anembodiment, a cell or a cell culture or expanded cells wherein saidformulation has been introduced by transfection according to theinvention. The cells are in particular mammalian cells, preferably humancells. The cells may be dividing cells or non-dividing cells.

In a particular embodiment of the invention, the composition accordingto the invention comprises from 1 to 5, preferably at least two distinctcompounds of general formula (I), preferably of general formula (III),or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixturethereof, or an acceptable salt thereof.

In a particular embodiment of the invention, the at least one preferredcompound of general formula (I) as defined herein is one wherein: (i) Y¹and Y³ represent N, Y² represents C; or (ii) Y¹ and Y² represent N, Y³represents C; or (iii) Y² and Y³ represent N, Y¹ represents C; or (iv)Y¹, Y² and Y³ represent N. When (iv) Y¹, Y² and Y³ represent N, the atleast one preferred compound of general formula (I) as defined hereincorresponds to the compound of general formula (III).

The structure of the compounds of general formula (III) is symmetric sothat R and V may be interchanged, and Z¹ and Z³ may be interchanged.Thus the definitions directed to R also apply to V, and the definitionsdirected to Z¹ also apply to Z³.

In a particular embodiment of the invention, the at least one preferredcompound of general formula (I), preferably of general formula (III), asdefined herein is one wherein (i) Z¹ represents H; or (ii) Z¹ representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein;more preferably Z¹ represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂represents CO, and R₃ represents (CH₂)_(m), with m representing aninteger between 1 and 3, preferably m is equal to 2.

In a particular embodiment of the invention, the at least one preferredcompound of general formula (I), preferably of general formula (III), asdefined herein is one wherein (i) Z² represents H, a C₁-C₁₂ alkoxy, or alinear or branched, saturated or unsaturated C₁-C₁₈ alkyl, preferably alinear or branched, saturated or unsaturated C₁-C₆ alkyl; morepreferably Z² represents H, CH₃, CF₃ or OCH₃; even more preferably Z²represents CH₃; or (ii) Z² represents X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺,R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺, preferably X₁—R₃—X₂—P⁺, wherein X₁,X₂, R₃ and P⁺ are as defined herein; more preferably Z² representsX₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂ represents CO, and R₃represents (CH₂)_(m), with m representing an integer between 1 and 3,preferably m is equal to 2.

In a particular embodiment of the invention, the at least one preferredcompound of general formula (I), preferably of general formula (III), asdefined herein is one wherein (i) Z³ represents H, a linear or branched,saturated or unsaturated C₁-C₁₈ alkyl, preferably a linear or branched,saturated or unsaturated C₁-C₆ alkyl, or a linear or branched, saturatedor unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, preferably fluorobenzyl or4-hydroxyphenethyl; or (ii) Z³ represents X₁—R₃—X₂—P⁺, X₁—R₃—P⁺,X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺, preferably X₁—R₃—X₂—P⁺,wherein X₁, X₂, R₃ and P⁺ are as defined herein; more preferably Z³represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂ represents CO, andR₃ represents (CH₂)_(m), with m representing an integer between 1 and 3,preferably m is equal to 2.

In a preferred embodiment of the invention, if (i) Z¹ representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein;more preferably Z¹ represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂represents CO, and R₃ represents (CH₂)_(m), with m representing aninteger between 1 and 3, preferably m is equal to 2 then (ii) Z²represents H, a C₁-C₁₂ alkoxy, or a linear or branched, saturated orunsaturated C₁-C₁₈ alkyl, preferably a linear or branched, saturated orunsaturated C₁-C₆ alkyl; more preferably Z² represents H, CH₃, CF₃ orOCH₃; and/or (iii) Z³ represents H, a linear or branched, saturated orunsaturated C₁-C₁₈ alkyl, preferably a linear or branched, saturated orunsaturated C₁-C₆ alkyl, or a linear or branched, saturated orunsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, preferably fluorobenzyl or4-hydroxyphenethyl; and/or (iv) R or V represents H, a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl or cycloalkyl, a C₆-C₁₈aryl, a linear or branched, saturated or unsaturated C₆-C₁₈ aryl-C₁-C₁₈alkyl, a linear or branched, saturated or unsaturated C₂-C₁₈heteroalkyl, a linear or branched, saturated or unsaturated C₁-C₂₄ester, a C₅-C₁₀ heterocyclyl, a C₅-C₁₀ heteroaryl, or a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl-C₅-C₁₀ heteroaryl.

In another preferred embodiment of the invention, if (i) Z² representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein;more preferably Z² represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂represents CO, and R₃ represents (CH₂)_(m), with m representing aninteger between 1 and 3, preferably m is equal to 2 then (ii) Z¹represents H; and/or (iii) Z³ represents H, a linear or branched,saturated or unsaturated C₁-C₁₈ alkyl, preferably a linear or branched,saturated or unsaturated C₁-C₆ alkyl, or a linear or branched, saturatedor unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, preferably fluorobenzyl or4-hydroxyphenethyl; and/or (iv) R or V represents H, a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl or cycloalkyl, a C₆-C₁₈aryl, a linear or branched, saturated or unsaturated C₆-C₁₈ aryl-C₁-C₁₈alkyl, a linear or branched, saturated or unsaturated C₂-C₁₈heteroalkyl, a linear or branched, saturated or unsaturated C₁-C₂₄ester, a C₅-C₁₀ heterocyclyl, a C₅-C₁₀ heteroaryl, or a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl-C₅-C₁₀ heteroaryl.

In another preferred embodiment of the invention, if (i) Z³ representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein;more preferably Z³ represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂represents CO, and R₃ represents (CH₂)_(m), with m representing aninteger between 1 and 3, preferably m is equal to 2 then (ii) Z¹represents H; and/or (iii) Z² represents H, a C₁-C₁₂ alkoxy, or a linearor branched, saturated or unsaturated C₁-C₁₈ alkyl, preferably a linearor branched, saturated or unsaturated C₁-C₆ alkyl; more preferably Z²represents H, CH₃, CF₃ or OCH₃; and/or (iv) R or V represents H, alinear or branched, saturated or unsaturated C₁-C₁₈ alkyl or cycloalkyl,a C₆-C₁₈ aryl, a linear or branched, saturated or unsaturated C₆-C₁₈aryl-C₁-C₁₈ alkyl, a linear or branched, saturated or unsaturated C₂-C₁₈heteroalkyl, a linear or branched, saturated or unsaturated C₁-C₂₄ester, a C₅-C₁₀ heterocyclyl, a C₅-C₁₀ heteroaryl, or a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl-C₅-C₁₀ heteroaryl.

In another preferred embodiment of the invention, if (i) R or Vrepresents X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, orX₂—P⁺, preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as definedherein; more preferably Z³ represents X₁—R₃—X₂—P⁺, wherein X₁ representsCH₂, X₂ represents CO, and R₃ represents (CH₂)_(m), with m representingan integer between 1 and 3, preferably m is equal to 2 then (ii) Z¹represents H; and/or (iii) Z² represents H, a C₁-C₁₂ alkoxy, or a linearor branched, saturated or unsaturated C₁-C₁₈ alkyl, preferably a linearor branched, saturated or unsaturated C₁-C₆ alkyl; and/or (iv) Z³represents H, a linear or branched, saturated or unsaturated C₁-C₁₈alkyl, preferably a linear or branched, saturated or unsaturated C₁-C₆alkyl, or a linear or branched, saturated or unsaturated C₆-C₁₈aryl-C₁-C₁₈ alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl.

In a particular embodiment of the invention, the at least one preferredcompound of general formula (I), preferably of general formula (III), asdefined herein is one wherein: (i) only one of Z¹, Z² or Z³ representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein;more preferably only one of Z¹, Z² or Z³ represents X₁—R₃—X₂—P⁺, whereinX₁ represents CH₂, X₂ represents CO, and R₃ represents (CH₂)_(m), with mrepresenting an integer between 1 and 3, preferably m is equal to 2;and/or (ii) Z¹ represents H; and/or (iii) Z² represents H, a C₁-C₁₂alkoxy, or a linear or branched, saturated or unsaturated C₁-C₁₈ alkyl,preferably a linear or branched, saturated or unsaturated C₁-C₆ alkyl;more preferably Z² represents H, CH₃, CF₃ or OCH₃; and/or (iv) Z³represents H, or a linear or branched, saturated or unsaturated C₁-C₁₈alkyl, preferably a linear or branched, saturated or unsaturated C₁-C₆alkyl.

In a particular embodiment of the invention, the at least one preferredcompound of general formula (I), preferably of general formula (III), asdefined herein is one wherein: if (i) R or V represents X₁—R₃—X₂—P⁺,X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺, preferablyX₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein; morepreferably Z³ represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂represents CO, and R₃ represents (CH₂)_(m), with m representing aninteger between 1 and 3, preferably m is equal to 2 then (ii) Z³ ispresent and Z³ represents H, a linear or branched, saturated orunsaturated C₁-C₁₈ alkyl, preferably a linear or branched, saturated orunsaturated C₁-C₆ alkyl, or a linear or branched, saturated orunsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, preferably fluorobenzyl or4-hydroxyphenethyl.

In a particular embodiment of the invention, the at least one preferredcompound of general formula (I), preferably of general formula (III), asdefined is one wherein the graft cationic polymer is selected from thegroup consisting of a linear or branched polyethyleneimine (PEI), PEIdendrimers, a polypropyleneimine (PPI), Poly(amidoamine) (PAA) anddendrimers (PAMAM), cationic cyclodextrin, polyalkylamine, apolyhydroxyalkylamine, poly(butyleneimine) (PBI), spermine, aN-substituted polyallylamine, N-substituted chitosan, a N-substitutedpolyornithine, a N-substituted polylysine (PLL), a N-substitutedpolyvinylamine, poly(β-amino ester), hyperbranched poly(amino ester)(h-PAE), networked poly(amino ester) (n-PAE), poly(4-hydroxy-1-prolineester) (PHP-ester) and a poly-β-aminoacid. Preferably the graft cationicpolymer is a linear or branched PEI, more preferably a linear PEI.

The graft cationic polymer may have a grafting ratio ranging from 1 to50%, preferably from 5 to 30%, more preferably is 20%.

As defined herein, the term “grafting ratio” refers to the number ofgrafted monomers on primary or secondary amino groups by side chains,divided by the number of total monomers present in the original cationicpolymer. The grafting ratio will depend upon the molecular weight of thecationic polymer, the chemical reactivity of the grafted side chainsonto the polymer, or the obtained biological effect. Said grafting ratiomay be determined by a measurement method well known in the art, forexample by NMR.

The graft cationic polymer may have an average molecular weight (Mw)ranging from 1 kDa to 500 kDa, preferably from 1 kDa to 50 kDa, morepreferably from 5 kDa to 50 kDa or from 1 kDa to 15 kDa. In particularthe graft cationic polymer may have an average molecular weight (Mw) of6, 8, 10, 15, 22 or 30 kDa, preferably of 6, 8, 10, 15 or 30 kDa.

The graft cationic polymer can be associated with a counterion such aschloride, phosphate, citrate, acetate, propionate, carbonate, succinate,sulfonate, sulfate, or carboxylate.

In a particular embodiment of the invention, the at least one preferredcompound of general formula (I), preferably of general formula (III), asdefined herein is one wherein Y¹, Y², Y³, Z¹, Z², Z³, X₁, X₂, R₃ and P⁺are as defined herein; and R or V represents H, a linear or branched,saturated or unsaturated C₁-C₁₈ alkyl or cycloalkyl, a C₆-C₁₈ aryl, alinear or branched, saturated or unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, alinear or branched, saturated or unsaturated C₂-C₁₈ heteroalkyl, alinear or branched, saturated or unsaturated C₁-C₂₄ ester, a C₅-C₁₀heterocyclyl, a C₅-C₁₀ heteroaryl, a linear or branched, saturated orunsaturated C₁₀-8 alkyl-C₅-C₁₀ heteroaryl, X₁—R₃—X₂—P⁺, X₁—R₃—P⁺,X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺.

Preferably, R or V represents H, methyl, ethyl, propyl, cyclopropyl,isopropyl, sec-butyl, cyclopentyl, phenyl, fluorophenyl, benzyl,pyridine, 2-pyridine, 3-pyridine, fluorobenzyl, substituted morpholinyl,substituted piperazinyl, 4-hydroxybenzyl, or 4-hydroxyphenethyl; morepreferably R or V represents methyl, ethyl, propyl, cyclopropyl,isopropyl, sec-butyl, cyclopentyl, phenyl, benzyl, fluorobenzyl,4-hydroxyphenethyl, 2-pyridine or 3-pyridine.

The most preferred embodiments for compounds of formula (III) inrelation to Z¹, Z², Z³, X₁, X₂, R₃ and P are as defined herein forcompounds of formula (I).

In a particular embodiment of the invention, preferred compounds arethose wherein only one of Z¹, Z² or Z³, preferably Z¹, representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined informula (I), preferably in formula (III).

In a particular embodiment of the invention, preferred compounds arethose wherein only one of R or V represents X₁—R₃—X₂—P⁺, X₁—R₃—P⁺,X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺, preferably X₁—R₃—X₂—P⁺,wherein X₁, X₂, R₃ and P⁺ are as defined in formula (I), preferably informula (III).

In a particular embodiment of the invention, preferred compounds arethose wherein Y¹, Y² and Y³ represent N. These compounds correspond tocompounds of general formula (III).

In a particular embodiment of the invention, preferred compounds arethose wherein Z³ represents a linear or branched, saturated orunsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, preferably fluorobenzyl or4-hydroxyphenethyl.

In a particular embodiment of the invention, preferred compounds arethose wherein R represents H, methyl, propyl, isopropyl, cyclopropyl,benzyl, fluorobenzyl, pyridine, 2-pyridine, 3-pyridine, phenyl,fluorophenyl, substituted morpholinyl or substituted piperazinyl.

In a particular embodiment of the invention, preferred compounds arethose wherein V represents H, X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺,X₁—P⁺, R₃—P⁺, or X₂—P⁺, preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ andP⁺ are as defined in formula (I), preferably in formula (III).

In a preferred embodiment of the invention, preferred compounds arethose wherein (i) Y¹, Y² and Y³ represent N; and/or (ii) V representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined informula (I); and/or (iii) R represents H; and/or (iv) Z³ representsfluorobenzyl or 4-hydroxyphenethyl.

In a preferred embodiment of the invention, preferred compounds arethose wherein (i) Y¹, Y² and Y³ represent N; and/or (ii) Z¹ representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₁—P⁺,preferably X₁—R₃—X₂—P⁺; and/or (iii) V represents H; and/or (iv) Rrepresents benzyl, fluorobenzyl, pyridine, 2-pyridine, 3-pyridine,methyl, propyl, isopropyl, cyclopropyl, phenyl, fluorophenyl,substituted morpholinyl or substituted piperazinyl.

According to a particular embodiment of the invention, preferredcompounds correspond to compounds 2.19 to 2.61, preferably compounds2.19, 2.22, 2.23, 2.42, 2.43, 2.44, 2.46, 2.47, 2.54, 2.55, 2.56, 2.57,2.58, 2.59, 2.60 and 2.61 as disclosed in Table 1.

TABLE 1 Structures of preferred compounds of the invention Com-Molecular Heterocycle pounds Structures weight Grafting (%) 2.19

22K 24% 2.20

22K 16% 2.21

22K 13% 2.22

22k 18% 2.23

22k 15% 2.24

22K 11% 2.25

22K 19% 2.26

22K 25% 2.27

22K  9% 2.28

22K 20% 2.29

22K  6% 2.30

22K 21% 2.31

22K 13% 2.32

22K  7% 2.33

22K 25% 2.34

22K 19% 2.35

22K 18% 2.36

22K 31% 2.37

22k 25% 2.38

22k 24% 2.39

22k 21% 2.40

22k 23% 2.41

22k 19% 2.42

22k 17% 2.43

22k 18% 2.44

10k 18% 2.45

22K  7% 2.46

10k 22% 2.47

10k 16% 2.48

25k 21% 2.49

10k 19% 2.50

15k 25% 2.51

22k 17% 2.52

25k 16% 2.53

22k 16% 2.54

22k 19% 2.55

22k 17% 2.56

22k 19% 2.57

22k 22% 2.58

22k 21% 2.59

22k 20% 2.60

22k 25% 2.61

22k 25%

In a particular embodiment of the invention, the at least one compoundof general formula (III) is selected from the group consisting of thefollowing compounds:

In a preferred embodiment of the invention, the at least one compound ofgeneral formula (III) is selected from the group consisting of thefollowing compounds: 2.19, 2.22, 2.42, 2.43, 2.44, 2.46, 2.47, 2.54,2.55, 2.56, 2.57, 2.58, 2.59, 2.60 and 2.61, even more preferably iscompound 2.22.

The at least one compound of general formula (I), preferably of generalformula (III), may be prepared according to various methods well knownin the art.

The present invention is also directed to the composition according tothe invention for use in in vivo applications for cell transformation byuptake of exogenous nucleic acid using the composition of the invention,for cell therapy or for gene therapy. The cells may be eukaryotic cells,in particular mammalian cells, especially human cells, in particularprimary cells, either dividing or non-dividing cells.

The present invention also concerns a method for in vitro or ex vivotransfection of live cells comprising introducing in the cells thecomposition according to the invention. Said live cells may be providedor maintained in medium containing serum, synthetic medium, animal-freecomponent medium or chemically defined medium.

The present invention also relates to the in vitro or ex vivo use of thecomposition according to the invention to transfect at least one nucleicacid molecule into a cell, cell line or cells, preferably a cell, cellline or cells selected from the group consisting of a mammalian cell, aninsect cell, a primary cell, an adherent cell, a suspension cell, adividing cell such as a stem cell, a non-dividing cell such as aneuronal cell, and a cancer cell, said cell, cell line or cells beingoptionally organized into spheroids, organoids, 2D or 3D cell culture,or provided as fibre or matrix culture, and/or within a bioreactor.

As defined herein, the term “adherent cells” refers to cells that needsolid support for growth, and are thus anchorage-dependent. Examples ofadherent cells include, but are not limited to, MRC-5 cells, HeLa cells,Vero cells, NIH-3T3 cells, L293 cells, CHO cells, BHK-21 cells, MCF-7cells, A549 cells, COS cells, HEK 293 cells, Hep G2 cells, SNN-BE (2)cells, BAE-1 cells or SH—SY5Y cells.

As defined herein, the term “suspension cells” refers to cells that donot need solid support for growth, and are thus anchorage-independent.Examples of suspension cells include, but are not limited to, NSO cells,U937 cells, Namalawa cells, HL60 cells, WEHI231 cells, Yac 1 cells,Jurkat cells, THP-1 cells, K562 cells or U266B1 cells.

As defined herein, the term “spheroids” refers to spherical,heterogenous aggregates of cells in culture that retainthree-dimensional architecture.

As defined herein, the term “organoids” refers to three-dimensionalstructures made of collection of organ-specific cell typesself-organized in a manner similar to in vivo.

As defined herein, the term “fibre or matrix culture” refers tothree-dimensional cell culture support composed of insoluble elasticfibers or extracellular proteins self-organized into matrix.

Said transfection may be stable or transient, standard or reverse.

As disclosed herein, the composition according to the invention maycomprise multiple distinct nucleic acids, in particular selected fromthe group consisting of multiple plasmid DNA, plasmid DNA andoligonucleotide, plasmid DNA and mRNA for co-transfection.

Said at least one nucleic acid molecule to be transfected may be a geneencoding a protein, a protein fragment, a peptide or an antibody orfunctional antigen-binding regions thereof, in particular VH and/or VLchains thereof. Said protein may be selected from the group consistingof a reporter protein, a fluorescent protein, an enzyme, a structuralprotein, a receptor, a transmembrane protein, a therapeutic protein, acytokine, a toxin, an oncogenic protein, an anti-oncogene, apro-apoptotic protein, an anti-apoptotic protein, a polymerase, atranscription factor and a capsid protein.

The present invention also relates to the in vitro or ex vivo use of thecomposition according to the invention for genome engineering, for cellreprogramming, in particular for the reprogramming of differentiatedcells into induced pluripotent stem cells (iPCs), for differentiatingcells, or for gene-editing. Such use may be carried out in a culture ofcells in vitro or ex vivo for the production of biologics, for thepreparation of cells for therapy purpose, or for the study of cellfunctions or behaviour in particular with a step of expansion of cellsafter their transfection or may be carried out in vivo for a therapeuticpurpose in a host in need thereof.

The present invention also relates to the in vitro or ex vivo use of thecomposition according to the invention (i) in the production ofbiologics, in particular biologics encoding a recombinant protein,peptide or antibody; or (ii) in the production of recombinant virus,such as adeno-associated virus (AAV), lentivirus (LV), adenovirus,oncolytic virus, or baculovirus, said composition comprising multiplenucleic acid molecules for co-transfection such as a plurality ofplasmids; or (iii) in the production of viral or virus-like particles,said composition comprising multiple nucleic acid molecules forco-transfection such as a plurality of plasmids.

Thus the present invention also relates to a method for the productionof (i) biologics, in particular biologics encoding a recombinantprotein, peptide or antibody; or (ii) recombinant virus, such asadeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolyticvirus, or baculovirus, wherein the composition according to theinvention comprises multiple nucleic acid molecules for co-transfection;or (iii) viral or virus-like particles, wherein the compositionaccording to the invention comprises multiple nucleic acid molecules forco-transfection.

In a preferred embodiment of the method for the production of AAV, saidcomposition comprises (i) at least one compound selected from the groupconsisting of compounds 2.22, 2.23, 2.43, 2.44, 2.47, 2.54, 2.57, 2.60and 2.61 and (ii) an acceptable excipient, buffering agent, cell culturemedium, or transfection medium.

In a preferred embodiment of the method for the production of LV, saidcomposition comprises (i) at least the compound 2.22, and (ii) anacceptable excipient, buffering agent, cell culture medium, ortransfection medium.

As defined herein, the term “biologics” refers to proteins or nucleicacids or combinations thereof, living entities such as cells or viruses,cell compartments, organoids, and tissues.

In a particular embodiment of the invention, said in vitro or ex vivouse of the composition or said method according to the invention is forthe production of recombinant virus, said composition comprising aplurality of expression vectors such as plasmid vectors to transfect inan adherent or suspension cell, such as HEK293 and derivative cells,HeLa, BHK-21, A549 or insect cells, wherein said vectors, in particularplasmids, are construct expressing viral structural sequences andtransfer vector genome for virus or virus-like production and optionallyexpressing molecules of interest encoded by the transfer vector genome.

In a particular embodiment of the invention, said recombinant virus isfor use in in vivo applications for cell therapy or for gene therapy.

In a particular embodiment of the invention, the invention relates tothe in vitro or ex vivo use of the composition according to theinvention in the production of a recombinant virus, such as anadeno-associated virus (AAV) or a lentivirus (LV), said compositioncomprising (i) at least one compound selected from the group ofcompounds 2.22, 2.23, 2.42, 2.43, 2.44, 2.46, 2.47, 2.54, 2.57, 2.60 and2.61 and (ii) an acceptable excipient, buffering agent, cell culturemedium, or transfection medium. Preferably, a composition comprising thecompound 2.22 is used in the production of LV; and a compositioncomprising at least one compound selected from the group consisting ofcompounds 2.22, 2.23, 2.43, 2.44, 2.47, 2.54, 2.57, 2.60 and 2.61 isused in the production of AAV.

Unless otherwise stated, all the above-mentioned embodiments may becombined together. Thus features which are described in the context ofseparate embodiments may be combined in a single embodiment.

Other features and advantages of the invention will be apparent from theexamples which follow and will also be illustrated in the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chemical structure of a compound of general formula (I).

FIG. 2. Percentage of GFP expression after transfection of Caco-2, HepG2, MDCK and MCF-10A with compounds of Example 3. The ratio 1:3 and 1:4indicate the ratio of μg of DNA per μL of compound.

FIG. 3. Production of AAV-2 from suspension HEK-293T cells. AAV-2vectors expressing the GFP reporter gene were produced in HEK-293T cellsgrown in suspension in FreeStyle F17 media. Cells were seeded andcultured for 3 days before being transfected by 3 plasmids (pAAV-RC2vector expressing Rep and Cap, pHelper vector expressing Adeno E2A,Adeno E4 and Adeno VA helper factors, and pAAV-GFP control vectorexpressing the GFP under the control of a CMV promoter) with PEIpro® orvarious compounds at ratio 1:2 or 1:3 μg DNA/μL reagent. AAV titers(transducing unit, TU/mL) were determined 72 hours post-transfection.The results are expressed as relative AAV-2 transducing Units/mL (TU/mL)in comparison to PEIpro® transfection at ratio 1:2 and 1:3.

FIG. 4. Production of lentivirus particles from suspension HEK-293Tcells. Lentivirus expressing the GFP reporter gene was produced inHEK-293T cells grown in suspension in FreeStyle F17 media. Cells wereseeded and cultured for 3 days before being transfected by 4 plasmidswith PEIpro® or compound 2.22 at ratio 1:2 μg total DNA/μL reagent.Lentivirus titers (transducing unit, TU/mL) were determined 72 hourspost-transfection.

FIG. 5. Chemical structure of a compound of general formula (III).

FIG. 6. Percentage of GFP expression after transfection of Hep G2 cellswith compounds 2.22 and 2.53 to 2.61. The ratio 1:3 and 1:4 indicate theratio of μg of DNA per μL of compound.

FIG. 7. Production of AAV-2 from suspension HEK-293T cells withcompounds 2.22 and 2.53 to 2.61. AAV-2 vectors expressing the GFPreporter gene were produced in HEK-293T cells grown in suspension inFreeStyle F17 media. Cells were seeded and cultured for 3 days beforebeing transfected by 3 plasmids (pAAV-RC2 vector expressing Rep and Cap,pHelper vector expressing Adeno E2A, Adeno E4 and Adeno VA helperfactors, and pAAV-GFP control vector expressing the GFP under thecontrol of a CMV promoter) with PEIpro® or various compounds at ratio1:2 μg DNA/μL reagent. AAV titers (transducing unit, TU/mL) weredetermined 72 hours post-transfection. The results are expressed asrelative AAV-2 transducing Units/mL (TU/mL).

FIG. 8. Influence of the amount of DNA transfected and the ratio ofcompound 2.22 per μg DNA on the production of AAV-2 from suspensionHEK-293T cells. AAV-2 vectors expressing the GFP reporter gene wereproduced in HEK-293T cells grown in suspension in FreeStyle F17 media.Cells were seeded and cultured for 3 days before being transfected by 3plasmids (pAAV-RC2 vector expressing Rep and Cap, pHelper vectorexpressing Adeno E2A, Adeno E4 and Adeno VA helper factors, and pAAV-GFPcontrol vector expressing the GFP under the control of a CMV promoter)with compound 2.22 (formulated at 15 mM nitrogen concentration) atdifferent ratio of μg DNA/μL reagent (ratio 1:1.5 to 1:3). AAV titers(transducing unit, TU/mL) were determined 72 hours post-transfection.The results are expressed as relative AAV-2 transducing Units/mL(TU/mL). The cell viability was determined 72 hours post-transfectionwith a Trypan blue assay.

FIG. 9. Influence of time of DNA complexation with compound 2.22 on theproduction of AAV-2 from suspension HEK-293T cells. AAV-2 vectorsexpressing the GFP reporter gene were produced in HEK-293T cells grownin suspension in FreeStyle F17 media. Cells were seeded and cultured for3 days before being transfected by 3 plasmids (pAAV-RC2 vectorexpressing Rep and Cap, pHelper vector expressing Adeno E2A, Adeno E4and Adeno VA helper factors, and pAAV-GFP control vector expressing theGFP under the control of a CMV promoter) with compound 2.22 (formulatedat 15 mM nitrogen concentration) at a ratio 1:2 of DNA/μL reagent andwith 1 μg DNA/million cells. AAV titers (transducing unit, TU/mL) weredetermined 72 hours post-transfection. The results are expressed asrelative AAV-2 transducing Units/mL (TU/mL).

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES Experimental SectionMaterial and Methods

Cell Culture

Caco-2 (ATCC® HTB-37™) human colon epithelial cells were grown in DMEM4.5 g/L glucose with 20% FBS supplemented with 1% non-essential aminoacids, 1 mM sodium pyruvate, 2 mM glutamine and 100 U/mL of penicillinand 100 μg/mL of streptomycin at 37° C. in a 5% CO₂ in air atmosphere.

MCF 10A (ATCC® CRL-10317™) human mammary epithelial cells were grown inMEBM (Lonza) supplemented with SingleQuots™ Supplements and GrowthFactors (Lonza) and 100 ng/ml cholera toxin at 37° C. in a 5% CO₂ in airatmosphere.

Hep G2 (ATCC® HB-8065™) human hepatocarcinoma cells were grown in MEM(Ozyme) with 10% FBS supplemented with 1% non-essential amino acids, 1mM sodium pyruvate, 2 mM glutamine and 100 U/mL of penicillin and 100μg/mL of streptomycin at 37° C. in a 5% CO₂ in air atmosphere.

MDCK (ATCC® CCL-34™) Madin-Darby canine kidney epithelial cells weregrown in MEM (Ozyme) with 10% FBS supplemented with 2 mM glutamine and100 U/mL of penicillin and 100 μg/mL of streptomycin at 37° C. in a 5%CO₂ in air atmosphere.

Primary human dermal fibroblasts were grown in DMEM (Ozyme) supplementedwith 10% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate, 2 mMglutamine and 100 U/mL of penicillin and 100 μg/mL of streptomycin at37° C. in a 5% CO₂ in air atmosphere.

Transfection Assay (96-Well Format)

One day before transfection, Caco-2, MCF 10A, Hep G2 and MDCK Cells wereseeded at 10 000, 25 000, 25 000, 10 000 cells per well (96-well plateformat), respectively, in 125 μL of their respective complete medium andincubated at 37° C. in a 5% CO₂ in air atmosphere. On the day oftransfection 200 ng of pCMV-EGFPLuc DNA (Clontech) was added in 20 μL ofOPTIMEM (Thermo Fisher), mixed with a vortex and incubated for 5 minutesat room temperature (rt). Then, 0.6 or 0.8 μL of a compound of generalformula (I), preferably of general formula (III) (at 7.5 mM nitrogenconcentration) were added onto the diluted DNA, mixed with a vortex andincubated for 10 minutes at rt. The transfection DNA solution (20 μL)was added into the well and the plate was incubated for 24 hours at 37°C. in a 5% CO₂ in air atmosphere.

For the GFP expression analysis, one day post-transfection, the cellculture medium was removed and 50 μL of trypsin-EDTA (1×, Lonza) wereadded per well and the plate was incubated for 5 minutes at 37° C. 150μL of complete medium were added to neutralize the trypsin, and the GFPexpression was analysed (2000 events) by flow cytometry (Exc 488 nm, Em520 nm) using a Guava easyCyte 6HT cytometer (Millipore).

Recombinant Virus Production

HEK-293T (ATCC® CRL-3216™): Human embryonic kidney cell is a highlytransfectable derivative of human embryonic kidney 293 cells, andcontains the SV40 T-antigen. HEK-293T cells are widely used forrecombinant virus production, gene expression and protein production.

For adherent cells, HEK-293T cells were seeded at 5×10⁶ cells in 145 cm²petri dishes in 15 mL of DMEM 4.5 g/L glucose supplemented with 10% FBS,2 mM glutamine and 100 U/mL of penicillin and 100 μg/mL of streptomycin,and incubated at 37° C. in a 5% CO₂ in air atmosphere.

AAV-2 was produced in HEK-293 T cells using the AAV-2 Helper FreePackaging System (catalog number VPK-402, Cell BIOLABS, INC.) byco-transfection of 3 plasmids, pAAV-RC2 vector expressing Rep and Cap,pHelper vector expressing Adeno E2A, Adeno E4 and Adeno VA helperfactors, and pAAV-GFP control vector expressing the GFP under thecontrol of a CMV promoter. Transfection complexes (10 μg total DNA perpetri dish) were prepared with a ratio of 2:2:1 with pAAV-RC2, pHelperand pAAV-GFP, respectively. Plasmids were diluted in a total volume of1.5 mL of OPTIMEM. Then, 20 or 30 μL of compounds were added onto thediluted DNA, mixed with a vortex and incubated for 10 minutes at rt.Transfection complexes were added onto the cells and the plate wasincubated for 72 h at 37° C. in a 5% CO₂ in air atmosphere.

For suspension cells, HEK-293T cells were seeded at 1×10⁶ cells/mL in 27mL of FreeStyle F17 supplemented with 4% Glutamine, 100 U/mL ofpenicillin, 100 μg/mL of streptomycin and 0.1% Pluronic in 125 mL flaskErlenmeyer (Corning). Cells were incubated for 24 h at 37° C. in an 8%CO₂ in air atmosphere under agitation (130 rpm). Plasmids(pAAV-GFP-pAAV-RC2—pHelper at ratio 2:2:1) were diluted in 3 mL ofFreeStyle F17. Then, compounds were added onto the diluted DNA (ratio 2or 3 μL per μg of DNA), mixed with a vortex and incubated for 10 minutesat rt. Transfection complexes were added onto the cells (2 μg DNA per1×10⁶ cells) and the plate was incubated for 72 h at 37° C. in a 8% CO₂in air atmosphere under agitation (130 rpm).

Lentivirus particles were produced using the ViraSafe™ LentiviralPackaging System, Pantropic (Catalog Number VPK-20, CELL BIOLABS INC.)containing pRSV-REV packaging vector, pCgpV Packaging Vector andpCMV-VSV-G Envelop Vector. pLenti6.3/V5-GW/EmGFP Expression ControlVector was from Thermo Fisher.

HEK-293T cells were seeded at 1×10⁶ cells/mL in 27 mL of FreeStyle F17supplemented with 4% Glutamine, 100 U/mL of penicillin, 100 μg/mL ofstreptomycin and 0.1% Pluronic in 125 mL flask Erlenmeyer (Corning).Cells were incubated for 24 h at 37° C. in an 8% CO₂ in air atmosphereunder agitation (130 rpm). Plasmids (pRSV-REV-pCgpV-pCMV-VSV-G-pLenti6.3at ratio 1:1:1:3) were diluted in 3 mL of FreeStyle F17. Then, compoundswere added onto the diluted DNA (ratio 2 μL per μg of DNA), mixed with avortex and incubated for 10 minutes at rt. Transfection complexes wereadded onto the cells (2 μg DNA per 1×10⁶ cells) and the plate wasincubated for 72 h at 37° C. in an 8% CO₂ in air atmosphere underagitation (130 rpm).

The transducing unit (TU/mL) was determined by using virus vectorsexpressing the GFP reporter gene after infection of permissive HT 1080cells for lentivirus vectors and HEK-293T cells for AAV-2 vectors in96-well and in presence of polybrene (8 μg/mL). The GFP expression wasanalysed by cytometry 72 h after transduction to determine thetransducing units.

Example 1. General Procedure for the Preparation of Grafted PolymersStep 1: Grafting

In a round-bottom flask was added the cationic polymer (1 equiv.) inwater (4 mL/mmol of starting material) followed by N-methyl morpholineor NMM (2 equiv.). The carboxylate (0.3-1 equiv.) was added followed byMeOH (16 mL/mmol of polymer). After stirring 10 min,4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride wasadded or DMTMM (0.6-2 equiv.) and the mixture was stirred 12-24 h atroom temperature. Then, MeOH was removed in vacuo, water (4 mL/mmol ofstarting material) followed by a solution of 3M HCl (1 mL/mmol ofstarting material) were added. The residue was purified using a dialysiscassette in a 50 mM HCl bath.

Step 2: Synthesis of Triazole by «Click» Chemistry Starting from an Acid

Alkyne (1 equiv.), azide (1 equiv.), CuSO₄ (0.01 equiv) and sodiumascorbate (0.03 equiv) were added to a 2:1 (v/v) solution of nBuOH andwater. The reaction was stirred at room temperature for 24 h. Then, NaOH(5M, 2 equiv.) was added and the organic solvent was removed in vacuo.The residue was purified by reversed phase flash chromatography using 0to 100% CH₃CN in water as eluant.

Step 3: Synthesis of Triazole by «Click» Chemistry Starting from anEster

Alkyne (1 equiv.), azide (1 equiv.), CuSO₄ (0.01 equiv) and sodiumascorbate (0.03 equiv) were added to a 2:1 (v/v) solution of nBuOH andwater. The reaction was stirred at room temperature for 24 h. Then, NaOH(5M, 2 equiv.) was added and the organic solvent was removed in vacuo.The residue was purified by reversed phase flash chromatography using 0to 100% CH₃CN in water as eluant.

Step 4: Saponification of the Ester Moiety

To a solution of ester in EtOH was added dropwise a 3M solution of LiOH,and the mixture was stirred at rt for the week-end. Then, the solventwas removed in vacuo and the residue was purified by reverse phase FC onSiO₂ using H₂O/MeCN as eluant using a Biotage Flash purification system.The acid obtained was lyophilized to yield a solid.

Step 5: Synthesis of Triazole by Ruthenium Catalyzed «Click» ChemistryStarting from an Ester

Cp*RuCl (cod) was added to a microwave vial. The vial was then evacuatedand backfilled with Argon (3×). Alkyne (1.1 eq.); alcyne (1 eq.) andtoluene were added to the vial under Ar and the mixture was stirred atrt overnight. Toluene was evaporated and the product was purified onreverse phase chromatography using H2O and MeCN.

The ester was retaken in EtOH and NaOH 1M (1.1 eq.) and stirred untilcompletion (followed by HPLC). EtOH was evaporated and the product waspurified by reverse phase chromatography using H2O and MeCN. The productwas lyophilized.

Step 6: Synthesis of 1,2,3-triazole

Triazole and K₂CO₃ in MeCN at 80° C. Add R—Br dropwise and stirred at80° C. overnight. Filtrate and washed the solid with MeCN. The filtratewas evaporated and purified by reverse phase chromatography (H₂O:MeCN).Two fractions were collected.

The esters were retaken in EtOH and NaOH 1M (1.1 eq) and stirred untilcompletion. EtOH was evaporated and the product was purified by reversephase chromatography H₂O:MeCN.

Example 2. Syntheses of Compounds of the Invention Synthesis of Product2.19

Intermediate 2.19a was prepared analogously to the general procedure,step 2 (Example 1). Yield=87%; m=520 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 7.75 (s, 1H), 7.34-7.25 (m, 2H), 7.18-7.04 (m, 2H), 5.50 (s,2H), 2.65 (t, J=7.2 Hz, 2H), 2.17 (t, J=7.1 Hz, 2H), 1.55 (dq, J=23.6,7.8 Hz, 3H).

Product 2.19 was prepared analogously to the general procedure, step 1(Example 1). Yield=36%; m=21 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.12-6.41 (m, 5H), 5.68-4.93 (m, 2H), 4.05-2.88 (m, 17H), 2.79-0.87 (m,8H).

Synthesis of Product 2.20

Intermediate 2.20a was prepared analogously to the general procedure,step 2 (Example 1). Yield=51%; m=261 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 7.75 (s, 1H), 7.34-7.25 (m, 2H), 7.18-7.04 (m, 2H), 5.50 (s,2H), 2.65 (t, J=7.2 Hz, 2H), 2.17 (t, J=7.1 Hz, 2H), 1.64-1.45 (m, 3H).

Product 2.20 was prepared analogously to the general procedure, step 1(Example 1). Yield=71%; m=31 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.17-6.68 (m, 5H), 5.60-5.28 (m, 2H), 4.10-2.93 (m, 27H).

Synthesis of Product 2.21

Intermediate 2.21a was prepared analogously to the general procedure,step 2 (Example 1). Yield=27%; m=148 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 7.71 (s, 1H), 7.32-7.24 (m, 2H), 7.09 (td, J=8.8, 2.0 Hz, 2H),5.55-5.46 (m, 2H), 2.86 (t, J=7.5 Hz, 2H), 2.45 (t, J=7.5 Hz, 2H).

Product 2.21 was prepared analogously to the general procedure, step 1(Example 1). Yield=29%; m=12 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.90-6.37 (m, 5H), 5.58-5.25 (m, 2H), 4.20-2.91 (m, 36H).

Synthesis of Product 2.22

Intermediate 2.22a was prepared analogously to the general procedure,step 2 (Example 1). Yield=28%; m=78 mg; ¹H NMR (400 MHz, Methanol-d₄) δ7.57 (s, 1H), 6.93-6.84 (m, 2H), 6.68-6.60 (m, 2H), 4.54-4.45 (m, 2H),3.04 (t, J=7.4 Hz, 2H), 2.99-2.91 (m, 2H), 2.53-2.44 (m, 2H).

Product 2.22 was prepared analogously to the general procedure, step 1(Example 1). Yield=87%; m=44 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ7.82-6.34 (m, 5H), 4.60-4.06 (m, 2H), 4.00-3.07 (m, 22H), 3.06-2.24 (m,7H).

Synthesis of Product 2.23

Intermediate 2.23a was prepared analogously to the general procedure,step 2 (Example 1). Yield=87%; m=258 mg; ¹H NMR (400 MHz, Methanol-d₄) δ7.53 (s, 1H), 6.98-6.87 (m, 2H), 6.75-6.63 (m, 2H), 4.53 (t, J=7.1 Hz,2H), 3.08 (t, J=7.1 Hz, 2H), 2.69 (t, J=7.6 Hz, 2H), 2.26-2.16 (m, 2H),1.91 (tt, J=8.3, 6.9 Hz, 2H).

Product 2.23 was prepared analogously to the general procedure, step 1(Example 1). Yield=100%; m=48 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.14-6.01 (m, 5H), 4.62-4.11 (m, 2H), 3.99-2.76 (m, 26H), 2.73-0.92 (m,8H).

Synthesis of Product 2.24

Intermediate 2.24a was prepared analogously to the general procedure,step 2 (Example 1). Yield=67%; m=379 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 7.62 (s, 1H), 7.24-7.14 (m, 2H), 7.05-6.92 (m, 2H), 5.35 (s,2H), 2.54 (t, J=7.6 Hz, 2H), 2.08 (t, J=7.5 Hz, 2H), 1.73 (tt, J 8.2,7.0 Hz, 2H).

Product 2.24 was prepared analogously to the general procedure, step 1(Example 1). Yield=97%; m=42 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ7.90-6.74 (m, 5H), 5.57-5.15 (m, 2H), 4.19-3.11 (m, 35H), 2.91-1.47 (m,6H).

Synthesis of Product 2.25

Product 2.25 was prepared analogously to the general procedure, step 1(Example 1). Yield=85%; m=41 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ7.84-6.46 (m, 5H), 5.54-4.94 (m, 2H), 4.15-3.11 (m, 26H), 2.97-1.11 (m,8H).

Synthesis of Product 2.26

Product 2.26 was prepared analogously to the general procedure, step 1(Example 1). Yield=80%; m=44 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.00-6.36 (m, 5H), 5.60-4.93 (m, 2H), 4.12-3.01 (m, 19H), 2.79-0.93 (m,8H).

Synthesis of Product 2.27

Intermediate 2.27a was prepared analogously to the general procedure,step 3 (Example 1). Yield=65%; m=305 mg; ¹H NMR (400 MHz, Chloroform-d)δ 8.03 (s, 1H), 8.00-7.91 (m, 2H), 7.31-7.21 (m, 2H), 5.36 (s, 2H), 3.96(s, 3H).

Intermediate 2.27b was prepared analogously to the general procedure,step 4 (Example 1). Yield=35%; m=97 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.07 (s, OH), 7.71-7.59 (m, 1H), 7.19-7.04 (m, 1H), 4.96 (s,1H).

Product 2.27 was prepared analogously to the general procedure, step 1(Example 1). Yield=67%; m=28 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ9.33-7.35 (m, 5H), 6.13-5.19 (m, 2H), 4.17-3.22 (m, 42H).

Synthesis of Product 2.28

Intermediate 2.28a was prepared analogously to the general procedure,step 3 (Example 1). Yield=62%; m=272 mg; ¹H NMR (400 MHz, Methanol-d₄) δ8.59 (d, J=5.0 Hz, 1H), 8.50 (s, 1H), 8.10 (dt, J=7.9, 1.1 Hz, 1H), 7.93(td, J=7.8, 1.8 Hz, 1H), 7.38 (ddd, J=7.6, 4.9, 1.2 Hz, 1H), 5.45 (s,2H), 3.83 (s, 3H).

Intermediate 2.28b was prepared analogously to the general procedure,step 4 (Example 1). Yield=94%; m=236 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.44-8.38 (m, 1H), 8.23 (s, 1H), 7.89-7.74 (m, 2H), 7.31 (ddd,J=6.0, 5.0, 2.8 Hz, 1H), 5.02 (s, 2H).

Product 2.28 was prepared analogously to the general procedure, step 1(Example 1). Yield=47%; m=23 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.06-6.11 (m, 5H), 5.55-4.96 (m, 2H), 4.26-2.20 (m, 20H).

Synthesis of Product 2.29

Intermediate 2.29a was prepared analogously to the general procedure,step 3 (Example 1). Yield=81%; m=355 mg; ¹H NMR (400 MHz, Methanol-d₄) δ9.08 (s, 1H), 8.64-8.46 (m, 2H), 8.28 (tt, J=6.3, 1.6 Hz, 1H), 7.55 (dd,J=8.0, 4.7 Hz, 1H), 5.44 (s, 2H), 3.84 (s, 2H).

Intermediate 2.29b was prepared analogously to the general procedure,step 4 (Example 1). Yield=88%; m=287 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.76 (dd, J=2.3, 0.9 Hz, 1H), 8.40 (dd, J=5.0, 1.6 Hz, 1H),8.23 (s, 1H), 8.07 (ddd, J=8.0, 2.3, 1.6 Hz, 1H), 7.42 (ddd, J=8.0, 5.0,0.9 Hz, 1H), 5.01 (s, 2H), 1.09 (t, J=7.1 Hz, 2H).

Product 2.29 was prepared analogously to the general procedure, step 1(Example 1). Yield=76%; m=29 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ9.28-7.21 (m, 5H), 5.94-5.16 (m, 2H), 4.19-2.35 (m, 19H).

Synthesis of Product 2.30

Product 2.30 was prepared analogously to the general procedure, step 1(Example 1). Yield=66%; m=32 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ9.47-7.87 (m, 5H), 6.08-5.50 (m, 2H), 4.32-2.94 (m, 50H).

Synthesis of Product 2.31

Intermediate 2.31a was prepared analogously to the general procedure,step 3 (Example 1). Yield=82%; m=354 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 9.46-7.68 (m, 5H), 6.03-5.32 (m, 2H), 4.28-2.83 (m, 50H). ¹HNMR (400 MHz, Methanol-d₄) δ 8.34 (s, 1H), 7.88-7.80 (m, 2H), 7.51-7.41(m, 2H), 7.41-7.32 (m, 1H), 5.39 (s, 2H), 3.83 (s, 3H).

Intermediate 2.31b was prepared analogously to the general procedure,step 4 (Example 1). Yield=99%; m=325 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.08 (s, 1H), 7.71-7.62 (m, 2H), 7.46-7.37 (m, 2H), 7.41-7.30(m, 1H), 4.94 (s, 2H).

Product 2.31 was prepared analogously to the general procedure, step 1(Example 1). Yield=58%; m=24 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.60-6.52 (m, 6H), 5.90-5.15 (m, 2H), 4.23-2.90 (m, 32H).

Synthesis of Product 2.32

Intermediate 2.32a was prepared analogously to the general procedure,step 3 (Example 1). Yield=87%; m=380 mg; ¹H NMR (400 MHz, Methanol-d₄) δ8.82-8.45 (m, 3H), 7.92 (s, 2H), 5.45 (s, 2H), 3.84 (s, 3H).

Intermediate 2.32b was prepared analogously to the general procedure,step 4 (Example 1). Yield=100%; m=351 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.49-8.43 (m, 2H), 8.34 (s, 1H), 7.70-7.64 (m, 2H), 5.02 (s,2H).

Product 2.32 was prepared analogously to the general procedure, step 1(Example 1). Yield=91%; m=32 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ9.17-8.00 (m, 5H), 6.13-5.23 (m, 2H), 4.21-3.01 (m, 74H).

Synthesis of Product 2.33

Intermediate 2.33a was prepared analogously to the general procedure,step 2 (Example 1). Yield=59%; m=49 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 7.90 (s, 1H), 4.40-4.32 (m, 2H), 3.69-3.62 (m, 6H), 2.52-2.45(m, 4H), 2.14-2.00 (m, 4H).

Product 2.33 was prepared analogously to the general procedure, step 1(Example 1). Yield=89%; m=15 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.63-7.84 (m, 1H), 4.52-4.23 (m, 3H), 4.13-2.86 (m, 27H), 2.74-1.54 (m,4H).

Synthesis of Product 2.34

Intermediate 2.34a was prepared analogously to the general procedure,step 2 (Example 1). Yield=48%; m=51 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 7.92 (s, 1H), 7.35-7.26 (m, 2H), 7.09-7.01 (m, 2H), 6.98 (tt,J=7.4, 1.1 Hz, 1H), 4.41-4.33 (m, 2H), 3.71 (s, 2H), 3.14-3.07 (m, 4H),2.69-2.61 (m, 4H), 2.14-2.00 (m, 4H).

Product 2.34 was prepared analogously to the general procedure, step 1(Example 1). Yield=98%; m=17 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.67-7.86 (m, 1H), 7.64-6.61 (m, 5H), 4.66-4.19 (m, 3H), 4.11-3.09 (m,31H), 2.80-1.74 (m, 4H).

Synthesis of Product 2.35

Intermediate 2.35a was prepared analogously to the general procedure,step 2 (Example 1). Yield=19%; m=20 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.24 (d, J=4.9 Hz, 2H), 7.95 (s, 1H), 6.64 (t, J=4.9 Hz, 1H),4.40-4.33 (m, 2H), 3.81-3.77 (m, 2H), 3.67-3.60 (m, 4H), 2.66-2.58 (m,4H), 2.12-2.00 (m, 4H).

Product 2.35 was prepared analogously to the general procedure, step 1(Example 1). Yield=44%; m=7 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.58-7.90 (m, 3H), 7.07-6.43 (m, 1H), 4.57-4.19 (m, 3H), 4.23-2.99 (m,32H), 2.83-1.68 (m, 4H).

Synthesis of Product 2.36

Intermediate 2.36a was prepared analogously to the general procedure,step 2 (Example 1). Yield=51%; m=78 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.39 (ddd, J=5.0, 1.7, 1.0 Hz, 1H), 8.21 (s, 1H), 7.85-7.71 (m,1H), 7.30 (ddd, J=7.3, 5.0, 1.5 Hz, 1H), 4.43-4.35 (m, 2H), 2.20-2.03(m, 4H).

Product 2.36 was prepared analogously to the general procedure, step 1(Example 1). Yield=77%; m=14 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ9.15-7.11 (m, 5H), 4.57-4.15 (m, 1H), 4.07-2.86 (m, 13H), 2.74-1.68 (m,4H).

Synthesis of Product 2.37

Intermediate 2.37a was prepared analogously to the general procedure,step 2 (Example 1). Yield=14%; m=38 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.00 (s, 1H), 7.57-7.47 (m, 2H), 7.09-6.95 (m, 2H), 4.26 (t,J=7.0 Hz, 2H), 2.08 (t, J=7.5 Hz, 2H), 1.76 (p, J=7.2 Hz, 2H), 1.49-1.35(m, 2H).

Product 2.37 was prepared analogously to the general procedure, step 1(Example 1). Yield=24%; m=9 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.23-6.04 (m, 5H), 4.39-2.72 (m, 18H), 2.70-0.56 (m, 6H).

Synthesis of Product 2.38

Intermediate 2.38a was prepared analogously to the general procedure,step 2 (Example 1). Yield=11%; m=27 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 7.97 (s, 1H), 7.51 (dd, J=8.7, 5.3 Hz, 2H), 7.02 (t, J=8.9 Hz,2H), 4.23 (t, J=7.1 Hz, 2H), 2.04 (t, J=7.5 Hz, 2H), 1.76 (p, J=7.2 Hz,2H), 1.45 (p, J=7.6 Hz, 2H), 1.21-1.09 (m, 2H).

Product 2.38 was prepared analogously to the general procedure, step 1(Example 1). Yield=18%; m=6 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.31-6.11 (m, 5H), 4.33-2.72 (m, 21H), 2.68-0.15 (m, 6H).

Synthesis of Product 2.39

Intermediate 2.39a was prepared analogously to the general procedure,step 2 (Example 1). Yield=35%; m=11 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.01 (s, 1H), 7.60-7.53 (m, 2H), 7.39-7.25 (m, 3H), 4.24 (t,J=7.1 Hz, 2H), 2.09 (t, J=7.5 Hz, 2H), 1.76 (p, J=7.2 Hz, 2H), 1.48-1.35(m, 2H).

Product 2.39 was prepared analogously to the general procedure, step 1(Example 1). Yield=53%; m=133 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.49-6.15 (m, 6H), 4.52-2.83 (m, 21H), 2.66-0.54 (m, 6H).

Synthesis of Product 2.40

Intermediate 2.40a was prepared analogously to the general procedure,step 2 (Example 1). Yield=76%; m=184 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 7.88 (s, 1H), 7.54-7.47 (m, 2H), 7.35-7.20 (m, 3H), 4.14 (t,J=7.1 Hz, 2H), 2.04 (t, J=7.5 Hz, 2H), 1.76-1.64 (m, 2H), 1.43 (p, J=7.6Hz, 2H), 1.21-1.06 (m, 2H).

Product 2.40 was prepared analogously to the general procedure, step 1(Example 1). Yield=12%; m=4 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ7.94-6.37 (m, 6H), 4.43-2.84 (m, 19H), 2.68-0.23 (m, 8H).

Synthesis of Product 2.41

Intermediate 2.41a was prepared analogously to the general procedure,step 2 (Example 1). Yield=26%; m=46 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 7.41 (s, 1H), 7.20-7.01 (m, 5H), 4.08 (t, J=7.0 Hz, 2H), 3.78(s, 2H), 2.00 (t, J=7.5 Hz, 2H), 1.72-1.50 (m, 2H), 1.32 (tt, J=15.0,9.9 Hz, 2H).

Product 2.41 was prepared analogously to the general procedure, step 1(Example 1). Yield=75%; m=26 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ7.99-6.38 (m, 6H), 4.32-1.58 (m, 30H).

Synthesis of Product 2.42

Intermediate 2.42a was prepared analogously to the general procedure,step 2 (Example 1). Yield=57%; m=96 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 7.15 (s, 1H), 7.05-6.86 (m, 5H), 3.92 (t, J=7.2 Hz, 2H), 3.67(s, 2H), 1.97 (t, J=7.6 Hz, 2H), 1.54-1.42 (m, 2H), 1.39-1.27 (m, 2H),1.05-0.92 (m, 2H).

Product 2.42 was prepared analogously to the general procedure, step 1(Example 1). Yield=87%; m=27 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ7.58-6.61 (m, 6H), 4.55-0.72 (m, 33H).

Synthesis of Product 2.43

Intermediate 2.43a was prepared analogously to the general procedure,step 2 (Example 1). Yield=24%; m=36 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.78 (s, 1H), 8.43-8.37 (m, 1H), 8.29 (s, 1H), 8.09 (dt, J=8.1,1.9 Hz, 1H), 7.43 (ddd, J=8.0, 5.0, 0.9 Hz, 1H), 4.38 (t, J=7.0 Hz, 2H),2.06 (t, J=7.5 Hz, 2H), 1.91-1.79 (m, 2H), 1.54-1.42 (m, 2H), 1.26-1.14(m, 2H).

Product 2.43 was prepared analogously to the general procedure, step 1(Example 1). Yield=38%; m=17 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ9.38-7.80 (m, 5H), 4.58-0.92 (m, 31H).

Synthesis of Product 2.44

Intermediate 2.44a was prepared analogously to the general procedure,step 2 (Example 1). Yield=25%; m=38 mg; ¹H NMR (400 MHz, DeuteriumOxide) δ 8.45-8.40 (m, 1H), 8.24 (s, 1H), 7.85-7.79 (m, 2H), 7.34-7.30(m, 1H), 4.37 (t, J=7.0 Hz, 2H), 2.05 (t, J=7.4 Hz, 2H), 1.91-1.79 (m,2H), 1.54-1.42 (m, 2H), 1.27-1.14 (m, 2H).

Product 2.44 was prepared analogously to the general procedure, step 1(Example 1). Yield=64%; m=29 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.79-7.46 (m, 5H), 4.56-2.83 (m, 23H), 2.72-0.78 (m, 8H).

Synthesis of Product 2.45

Product 2.45 was prepared analogously to the general procedure, step 1(Example 1). Yield=49%; m=18 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.01-6.82 (m, 5H), 5.62-5.23 (m, 2H), 4.04-3.18 (m, 66H), 2.95-1.63 (m,6H).

Synthesis of Product 2.46

Product 2.46 was prepared analogously to the general procedure, step 1(Example 1). Yield=93%; m=156 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.98-8.37 (m, 3H), 8.27 (s, 1H), 7.90 (s, 1H), 4.61-4.32 (m, 2H),4.05-3.13 (m, 18.5H), 2.56-2.18 (m, 2H), 2.12-1.79 (m, 2H), 1.73-1.42(m, 2H), 1.42-1.14 (m, 2H).

Synthesis of Product 2.47

Product 2.47 was prepared analogously to the general procedure, step 1(Example 1). Yield=99%; m=44 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ8.00-6.02 (m, 5H), 4.66-4.05 (m, 1H), 3.97-2.01 (m, 32H).

Synthesis of Product 2.48

Product 2.48 was prepared analogously to the general procedure, step 1.Yield=28%; m=83 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 7.75-7.23 (s,1H), 7.15-6.38 (m, 4H), 4.64-4.17 (m, 2H), 4.14-2.00 (m, 25H).

Synthesis of Product 2.49

Product 2.49 was prepared analogously to the general procedure, step 1.Yield=5%; m=13 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 7.78-7.32 (s,1H), 7.20-6.31 (m, 4H), 4.66-4.31 (m, 2H), 4.22-2.20 (m, 27H).

Synthesis of Product 2.50

Product 2.50 was prepared analogously to the general procedure, step 1.Yield=18%; m=29 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 7.71-7.19 (s,1H), 7.15-6.34 (m, 4H), 4.65-4.09 (m, 2H), 4.06-0.57 (m, 26H).

Synthesis of Product 2.51

Product 2.51 was prepared analogously to the general procedure, step 1.Yield=36%; m=13 mg ¹H NMR (400 MHz, Deuterium Oxide) δ 7.68-7.45 (m,1H), 7.14-6.62 (m, 4H), 4.67-4.42 (m, 2H), 4.39-4.17 (m, 5H), 3.38-2.73(m, 16H), 2.58-2.33 (m, 2H), 1.99-1.01 (m, 35H).

Synthesis of Product 2.52

Product 2.52 was prepared analogously to the general procedure, step 1.Yield=43%; m=63 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 7.84-7.31 (s,1H), 7.28-6.48 (m, 4H), 4.69-4.33 (m, 2H), 4.30-1.04 (m, 25H).

Synthesis of Product 2.53

Intermediate 2.53a was prepared analogously to the general procedure,steps 3 & 4. Yield=34%; m=151 mg ¹H NMR (400 MHz, MeOD) δ 6.69 (s, 1H),3.39-3.21 (m, 2H), 1.20-0.94 (m, 4H), 0.26 (s, 9H).

Product 2.53 was prepared analogously to the general procedure, step 1.Yield=37%; m=25 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 8.07-7.88 (m,1H), 4.60-3.35 (m, 2H), 4.08-3.05 (m, 24H), 2.90-1.97 (m, 4H), 1.55-1.05(m, 9H)

Synthesis of Product 2.54

Intermediate 2.54a was prepared analogously to the general procedure,step 3 & 4. Yield=69%; m=287 mg ¹H NMR (400 MHz, MeOD) δ 6.64 (s, 1H),3.32 (d, J=6.9 Hz, 2H), 1.17-1.00 (m, 4H), 0.90 (tt, J=8.4, 5.0 Hz, 1H),−0.02-−0.18 (m, 2H), −0.22-−0.35 (m, 2H).

product 2.54 was prepared analogously to the general procedure, step 1.Yield=34%; m=24 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 7.99-7.78 (m,1H), 4.61-4.38 (m, 2H), 4.06-3.26 (m, 21H), 2.66-1.95 (m, 5H), 1.15-1.01(m, 2H), 0.88-0.70 (m, 2H).

Synthesis of Product 2.55

Intermediate 2.55a was prepared analogously to the general procedure,step 3. Yield=52%; m=251 mg; ¹H NMR (500 MHz, CDCl₃) δ 7.28 (s, 1H),4.39 (t, J=6.9 Hz, 2H), 4.14 (q, J=7.1 Hz, 2H), 3.28-3.09 (m, 1H), 2.34(t, J=6.9 Hz, 2H), 2.22 (p, J=7.0 Hz, 2H), 2.11 (s, 2H), 1.88-1.56 (m,6H), 1.26 (t, J=7.1 Hz, 3H).

Intermediate 2.55b was prepared analogously to the general procedure,step 4. Yield=quantitative; m=223 mg; ¹H NMR (400 MHz, MeOD) δ 7.65 (s,1H), 4.35-4.21 (m, 2H), 3.12-2.96 (m, 1H), 2.15-1.91 (m, 6H), 1.77-1.49(m, 6H).

Product 2.55 was prepared analogously to the general procedure, step 1.Yield=40%; m=22 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 8.04-7.83 (m,1H), 4.59-4.34 (m, 2H), 4.07-3.04 (m, 25H), 2.60-1.97 (m, 6H), 1.80-1.47(m, 6H)

Synthesis of Product 2.56

Intermediate 2.56a was prepared analogously to the general procedure,step 3. Yield=80%; m=350 mg; ¹H NMR (400 MHz, CDCl₃) δ 7.26 (s, 1H),4.38 (t, J=6.9 Hz, 2H), 4.14 (q, J=7.1 Hz, 2H), 3.09 (hept, J=6.9 Hz,1H), 2.34 (dd, J=7.4, 6.4 Hz, 2H), 2.29-2.14 (m, 2H), 1.31 (s, 3H), 1.29(s, 3H), 1.26 (td, J=7.1, 0.6 Hz, 3H).

Intermediate 2.56b was prepared analogously to the general procedure,step 4. Yield=quantitative; m=340 mg; ¹H NMR (400 MHz, MeOD) δ 7.75 (d,J=0.8 Hz, 1H), 4.39 (td, J=6.4, 5.8, 2.8 Hz, 2H), 3.03 (pd, J=6.9, 0.7Hz, 1H), 2.25-2.05 (m, 4H), 1.30 (s, 3H), 1.29 (s, 3H).

Product 2.56 was prepared analogously to the general procedure, step 1.Yield=45%; m=32 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 8.09-7.90 (m,1H), 4.61-4.36 (m, 2H), 4.09-3.20 (m, 21H), 3.17-2.97 (m, 1H), 2.62-2.03(m, 4H), 1.45-1.16 (m, 6H).

Synthesis of Product 2.57

Intermediate 2.57a was prepared analogously to the general procedure,step 5. Yield=55%; m=287 mg; ¹H NMR (400 MHz, CDCl₃) δ 7.73-7.66 (m,2H), 7.56-7.40 (m, 4H), 7.38-7.27 (m, 2H), 4.39 (t, J=7.1 Hz, 2H), 4.31(t, J=6.9 Hz, 1H), 4.14 (q, J=7.1 Hz, 2H), 4.04 (q, J=7.1 Hz, 1H), 2.48(s, 3H), 2.43 (t, J=7.0 Hz, 2H), 2.30 (s, 2H), 2.31-2.23 (m, 1H),2.27-2.19 (m, 2H), 2.15-2.03 (m, 1H), 1.26 (t, J=7.1 Hz, 3H), 1.19 (t,J=7.1 Hz, 2H).

Intermediate 2.57b was prepared analogously to the general procedure,step 4. Yield=quantitative; m=280 mg; ¹H NMR (400 MHz, MeOD) δ 7.79-7.10(m, 5H), 4.56-4.17 (m, 2H), 2.57-1.89 (m, 7H).

Product 2.57 was prepared analogously to the general procedure, step 1.Yield=55%; m=44 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 7.68-6.86 (m,5H), 4.53-3.03 (m, 20H), 2.62-1.58 (m, 7H).

Synthesis of Product 2.58

Intermediate 2.58a was prepared analogously to the general procedure,step 5. Yield=49%; m=224 mg; ¹H NMR (400 MHz, CDCl₃) δ 4.35-4.25 (m,2H), 4.19-4.08 (m, 2H), 3.23-2.96 (m, 1H), 2.42-2.34 (m, 4H), 2.22-2.09(m, 2H), 1.36-1.30 (m, 6H), 1.29-1.18 (m, 3H).

Intermediate 2.58b was prepared analogously to the general procedure,step 4. Yield=quantitative; m=219 mg; ¹H NMR (400 MHz, MeOD) δ 4.37-4.27(m, 2H), 3.31-3.18 m 1H) 2.33 (s, 31H), 2.27-2.13 (m, 2H), 2.15-2.03 (m,2H), 1.34 (s, 3H), 1.32 (s, 3H).

Product 2.58 was prepared analogously to the general procedure, step 1.Yield=64%; m=48 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 4.49-4.22 (m,2H), 3.99-3.03 (m, 20H), 2.76-1.87 (m, 7H), 1.34-1.04 (m, 6H).

Synthesis of Product 2.59

Intermediate 2.59a was prepared analogously to the general procedure,step 3. Yield=41%; m=199 mg; ¹H NMR (400 MHz, CDCl₃) δ 7.25 (s, 1H),4.39 (t, J=6.9 Hz, 2H), 4.14 (q, J=7.1 Hz, 2H), 2.96 (h, J=7.0 Hz, 1H),2.34 (td, J=7.1, 1.0 Hz, 2H), 2.21 (p, J=7.0 Hz, 2H), 1.75-1.62 (m, 1H),1.60-1.46 (m, 1H), 1.41-1.16 (m, 8H), 0.90 (t, J=7.3 Hz, 3H).

Intermediate 2.59b was prepared analogously to the general procedure,step 4. Yield=quantitative; m=194 mg; ¹H NMR (400 MHz, MeOD) δ 7.75 (s,1H), 4.44-4.35 (m, 2H), 2.92 (h, J=7.0 Hz, 1H), 2.19-2.08 (m, 4H),1.74-1.49 (m, 2H), 1.47-1.10 (m, 6H), 0.91 (t, J=7.4 Hz, 3H).

Product 2.59 was prepared analogously to the general procedure, step 1.Yield=58%; m=44 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 7.98-7.77 (m,1H), 4.58-4.31 (m, 2H), 4.07-3.16 (m, 20H), 3.05-2.77 (m, 1H), 2.60-1.98(m, 4H), 1.66-1.42 (m, 2H), 1.34-1.04 (m, 5H), 0.92-0.64 (m, 3H).

Synthesis of Product 2.60

Intermediate 2.60a was prepared analogously to the general procedure,step 6. Yield=44%; m=586 mg; ¹H NMR (400 MHz, CDCl₃) δ 7.75-7.68 (m,1H), 7.58 (s, 1H), 4.48 (td, J=6.8, 1.9 Hz, 2H), 4.21-4.04 (m, 2H),2.43-2.29 (m, 2H), 2.29-2.19 (m, 2H), 1.33-1.18 (m, 3H).

Intermediate 2.60b was prepared analogously to the general procedure,step 4. Yield=quantitative; m=567 mg; ¹H NMR (400 MHz, MeOD) δ 8.01 (d,J=1.3 Hz, 1H), 7.71 (d, J=1.2 Hz, 1H), 4.52-4.43 (m, 2H), 2.23-2.10 (m,4H).

Product 2.60 was prepared analogously to the general procedure, step 1.Yield=46%; m=33 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 8.09-7.95 (m,1H), 7.89-7.76 (m, 1H) 4.59-4.41 (m, 2H), 4.04-3.12 (m, 16H), 2.55-1.99(m, 4H).

Synthesis of Product 2.61

Intermediate 2.61a was prepared analogously to the general procedure,step 6. Yield=27%; m=362 mg; ¹H NMR (400 MHz, CDCl₃) δ 7.59 (s, 2H),4.51 (td, J=6.6, 1.1 Hz, 2H), 4.13 (qd, J=7.1, 1.2 Hz, 2H), 2.36-2.26(m, 4H), 1.24 (td, J=7.1, 1.1 Hz, 3H).

Intermediate 2.61b was prepared analogously to the general procedure,step 4. Yield=quantitative; m=350 mg; ¹H NMR (400 MHz, MeOD) δ 7.54 (s,2H), 4.42-4.34 (m, 2H), 2.16-2.00 (m, 4H).

Product 2.61 was prepared analogously to the general procedure, step 1.Yield=41%; m=29 mg; ¹H NMR (400 MHz, Deuterium Oxide) δ 7.84-7.67 (m,2H), 4.60-4.39 (m, 2H), 4.08-3.12 (m, 16H), 2.57-1.99 (m, 4H).

Example 3. Compounds 2.19 to 2.26

Screening of Transfection Activity

Compounds 2.19 to 2.26 were evaluated for their ability to transfect DNAin four different cell lines, Caco-2 (human colon epithelial cells), HepG2 (human hepatocarcinoma cells), MDCK (Madin-Darby canine kidneyepithelial cells) and MCF-10A (human mammary epithelial cells). Thescreening of compounds (FIG. 2) was performed in 96-well plate bytransfecting 200 ng of pCMV-EGFPLuc DNA (Clontech) complexed with 0.6 or0.8 μL of one compound of the invention, i.e. one compound selected fromthe group consisting of compounds 2.19 to 2.26 (at 7.5 mM nitrogenconcentration), defining a ratio of 1 μg DNA/3 μL of compound (ratio1:3) or a ratio of 1 μg DNA/4 μL of compound (ratio 1:4), respectively.The percentage of cells expressing the GFP (% GFP) was determined bycytometry assay one day post-transfection. A transfection was performedwith jetPEI® as a control which is a linear polyethylenimine of 22 KDaand represents the parental cationic polymer backbone of the testedcompounds.

The compounds 2.19 to 2.26 represent polymers wherein the triazole ringwas used to graft fluorobenzyl or hydroxyphenol (or 4-hydroxyphenethyl)moiety and wherein the cationic polymer is grafted to R or V of theformula (III). All the compounds showed significant transfectionactivity whereas the best compound was dependent to the cell line used.

Example 4. Bioproduction of Recombinant Virus with Compounds 2.22, 2.23,2.41, 2.42, 2.43, 2.46 and 2.47

DNA transfection is one of the mainly used technologies in thebioproduction of recombinant proteins and viruses by a process oftransient gene expression (TGE). Concerning the production of AAV andlentivirus the most commonly used method is the transfection to deliverthe viral and therapeutic genes in the producer cell lines, HEK293adherent of suspension cells. In most systems, the co-transfection ofmany plasmids is performed by a chemical method, such as theco-precipitation with the calcium phosphate or the transfection mediatedwith the cationic polymer polyethylenimine (PEI), such as PEIpro®(Polyplus-transfection) commercially recommended for such abioproduction of recombinant virus.

AAV and lentivirus particles were produced from HEK-293T cells throughtransient co-transfection of several plasmids containing the gene ofinterest and necessary viral components to produce full recombinantvirions. AAV-2 and lentivirus vectors expressing the GFP reporter genewere produced with various compounds and the virus productivity wasdetermined by assessing the transducing unit (TU/mL) 3 dayspost-transfection. The levels of productivity were compared to thoseobtained with the PEIpro® transfection reagent extensively used inadherent and suspension virus production systems.

Many compounds of Example 3 were tested for the production of AAV-2 aswell as other compounds wherein the triazole ring was grafted by benzyl(2.41 or 2.42) or pyridinyl (2.43 to 2.46) moiety and wherein thecationic polymer was linked to the triazole ring in position Z¹ of theformula (III). FIG. 3 presents some of the results obtained. At a ratioof 1:2 (1 μg total DNA per μL of compound) used for the transfection,some compounds performed similarly in virus productivity than PEIpro®but most of them increased significantly by 3- to 8-fold the viraltiter. This improvement was confirmed for most of the compounds andenhanced by using a ratio of 1:3 with the highest increase of viraltiter superior to 10-fold for compound 2.43.

Similarly, lentiviruses were produced in suspension HEK-293T cells afterco-transfection of 4 plasmids (pRSV-REV packaging vector, pCgpVPackaging Vector, pCMV-VSV-G Envelop Vector and pLenti6.3/V5-GW/EmGFPExpression Control Vector). Lentivirus titers (TU/mL) were determined 72hours post-transfection (FIG. 4). An improvement of the LV productionyield of about 10-fold was obtained when compared to the productivitywith PEIpro® by using the compound 2.22 at a ratio of 1:3.

Example 5. Compounds 2.53 to 2.61

Screening of Transfection Activity

Compounds 2.53 to 2.61 were screened in transfection (FIG. 6) similarlyas previously described for compounds of Example 3, in 96-well plate bytransfecting 200 ng of pCMV-EGFPLuc DNA (Clontech) complexed with 0.6 or0.8 μL of one compound of the invention (at 7.5 mM nitrogenconcentration), defining a ratio of 1 μg DNA/3 μL of compound or ratioof 1 μg DNA/4 μL of compound, respectively.

Compounds 2.53 to 2.61 represent compounds having a triazole ringwherein the cationic polymer is linked at Z¹ of the formula (III) andwherein various alkyl or cycle moiety where added on position R or V ofthe formula (III). FIG. 6 shows that grafting of alkyl or cycloalkylmoiety at the position R or V on the triazole ring provides efficientcompounds in transfection as exemplified by the compounds 2.54, 2.56,2.58 or 2.57. Surprisingly, compounds 2.60 and 2.61 with unsubstitutedtriazole ring on position R and V of the formula (III) were not able totransfect efficiently the Hep G2 cells.

Bioproduction of Recombinant Virus

Compounds 2.53 to 2.61 were tested for the production of AAV-2 and FIG.7 presents the results obtained of compounds at ratio 1:2 μg DNA/μLreagent. AAV titers (transducing unit, TU/mL) were determined 72 hourspost-transfection. The results are expressed as relative AAV-2transducing Units/mL (TU/mL).

The compound 2.22 was used as a positive control. Compounds 2.54 and2.57 showed promising results and in correlation with the transfectionactivity presented in FIG. 6. Contrary to the experiments oftransfection in Hep G2 cells, compounds 2.60 and 2.61 wherein R and V═Hshowed high levels of AAV-2 productivity in HEK-293T cells

Example 6. Key Parameters for the Production of AAV-2 from SuspensionHEK-293T Cells

The production of recombinant virus is achieved by co-transfection ofmany plasmids in HEK293 cells. The virus productivity is greatlyinfluenced by the total amount of plasmids and the volume oftransfection reagent. FIG. 8 illustrates the AAV-2 production using thecompound 2.22 (formulated at 15 mM nitrogen concentration). Differentamounts of plasmids were used to transfect the HEK293-T cells insuspension. Many ratios of transfection reagent were also tested andexpressed as μg DNA/μL reagent per millions of cells the day oftransfection. The results show that the virus productivity depends onthe amount of plasmids transfected. In addition, for each amount of DNAtransfected, the optimal productivity depends on the ratio of μg DNA/μLreagent. This example illustrates the transfection conditions with thecompounds of formula (III) can be adapted easily to obtain an optimalvirus productivity. FIG. 9 presents the influence of time of DNAcomplexation with the compound 2.22 on the production of AAV-2 fromsuspension HEK-293T cells. A minimal time of DNA complexation of 15minutes before adding the transfection complexes into the cell cultureis required to obtained high yield of virus production. A longer time ofDNA complexation above 15 minutes can be used without affecting thevirus yield, indicating a good stability of the transfection complexesin virus production activity. This property indicates that the compound2.22 is particularly suitable for large scale applications inbioreactors where the time window during the transfer of thetransfection complexes mixture needs to be adapted according to the cellculture volume.

CONCLUSION

Many compounds based on grafting of polyamine with heterocycles offormula (I), preferably of formula (III) showed improved performances toinduce gene expression in “hard to transfect” cells such as cancercells, or to increase the productivity of biologics such as viruses, AAVor LV.

Many compounds of Example 3, 4 or 5, particularly polyamine grafted withbenzyl, fluorobenzyl, hydroxyphenyl, 4-hydroxyphenethyl, pyridine orphenyl triazole derivative showed high transfection efficiencies.

Selected compounds of Example 3, 4 or 5 also showed improvedproductivity of biologics such as AAV or LV, indicating a combinedeffect of high transfection efficiency and gene expression in cellsresulting in high virus titers expressed as transducing units. Improvedvirus productivity was observed whatever the type of transfected cells,e.g. adherent or in suspension. The results obtained indicated that suchcompounds might be also of interest to produce other biologics such asrecombinant proteins, peptides or antibodies.

Taken together, the compounds of formula (I), preferably of formula(III) of the invention represent novel reagents for transfection andbioproduction purposes wherein a fine optimisation of the chemicalstructure may be adapted for each application, cell types ortransfection conditions.

The person skilled in the art can adapt the transfection method with thecompounds of general formula (I), preferably of general formula (III) ofthe invention for in vivo applications with an acceptable excipient orbuffering agent. The compounds of general formula (I), preferably ofgeneral formula (III) can be mixed with DNA to generate DNA complexessuitable for direct injection into animals or humans. Particularly lowsalt buffering agents such as TRIS, phosphate, or citrate buffer orexcipient such as glucose, dextrose, or maltose are known to provideacceptable formulation for direct injection into animals and humans.Many mixture methods between the DNA and the compounds of generalformula (I), preferably of general formula (III) are suitable as theyare able to generate formulation containing small size particles (nonaggregated DNA complexes) that can be injected through various routes ofadministration.

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1. A composition suitable for transfecting a nucleic acid molecule intoa cell, preferably a eukaryotic cell, comprising (i) at least onecompound of general formula (III) or a tautomer, mesomer, racemate,enantiomer, diastereomer, or mixture thereof, or an acceptable saltthereof, and (ii) an acceptable excipient, buffering agent, cell culturemedium, or transfection medium:

wherein: Z¹ represents H, X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺,X₁—P⁺, R₃—P⁺, or X₂—P⁺; or Z¹ is absent; Z² represents H, a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl, C₆-C₁₈ aryl, a linearor branched, saturated or unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, a linearor branched, saturated or unsaturated C₂-C₁₈ heteroalkyl, C₅-C₁₀heteroaryl, halogen, OH, a linear or branched, saturated or unsaturatedC₁-C₁₈ alkylamine, a C₁-C₁₂ alkoxy, a linear or branched, saturated orunsaturated C₁-C₁₈ alkyl-C₁-C₁₂ alkoxy, X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺,R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺; or Z² is absent; Z³ represents H, alinear or branched, saturated or unsaturated C₁-C₁₈ alkyl, C₆-C₁₈ aryl,a linear or branched, saturated or unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl,C₅-C₁₀ heteroaryl, a linear or branched, saturated or unsaturated C₂-C₁₈heteroalkyl, C₂-C₁₈ alkylidene, OH, guanidine, halogen, X₁—R₃—X₂—P⁺,X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺; or Z³ is absent;X₁ and X₂, which may be identical or different, represent CO or CH₂; R₃represents (CH₂)_(m), (CH₂)_(m)—CHCH₃—(CH₂)_(n)—,(CH₂)_(m)—C(CH₃)₂—(CH₂)_(n)—, (CH₂)_(m)—O—(CH₂)_(n)—,(CH₂)_(m)—S—(CH₂)_(n)—, (CH₂)_(m)—CH₂—O—, with m representing an integerbetween 1 and 3 and n representing an integer between 1 and 3; P⁺represents a graft cationic polymer, which is a polyamine comprisingsecondary amines, tertiary amines, a mixture of primary and secondaryamines, a mixture of primary and tertiary amines, a mixture of secondaryand tertiary amines, or a mixture of primary, secondary and tertiaryamines; R or V represents H, a linear or branched, saturated orunsaturated C₁-C₁₈ alkyl or cycloalkyl, a C₆-C₁₈ aryl, a linear orbranched, saturated or unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, a linear orbranched, saturated or unsaturated C₂-C₁₈ heteroalkyl, a linear orbranched, saturated or unsaturated C₁-C₂₄ ester, a C₅-C₁₀ heterocyclyl,a C₅-C₁₀ heteroaryl, a linear or branched, saturated or unsaturatedC₁-C₁₈ alkyl-C₅-C₁₀ heteroaryl, X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺,R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺; with the provisos that: at least oneof Z¹, Z² or Z³ is present; and only one of Z¹, Z², Z³, R or Vrepresents X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, orX₂—P⁺.
 2. The composition according to claim 1, further comprising atleast one nucleic acid molecule to be transfected in a cell, preferablya nucleic acid molecule selected from the group consisting of adeoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a DNA/RNA hybrid,a short interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA(shRNA), a messenger RNA (mRNA), a CRISPR guide RNA, and an expressionvector encoding said nucleic acid molecule, in particular a plasmidencoding said nucleic acid molecule or a plasmid expressing said nucleicacid molecule.
 3. The composition according to claim 2, wherein the atleast one nucleic acid molecule is a DNA.
 4. The composition accordingto claim 1, wherein R or V represents H, methyl, ethyl, propyl,cyclopropyl, isopropyl, sec-butyl, cyclopentyl, phenyl, fluorophenyl,benzyl, pyridine, 2-pyridine, 3-pyridine, fluorobenzyl, substitutedmorpholinyl, substituted piperazinyl, 4-hydroxybenzyl, or4-hydroxyphenethyl; more preferably R or V represents methyl, ethyl,propyl, cyclopropyl, isopropyl, sec-butyl, cyclopentyl, phenyl, benzyl,fluorobenzyl, 4-hydroxyphenethyl, 2-pyridine or 3-pyridine.
 5. Thecomposition according to claim 1, wherein: (i) only one of Z¹, Z² or Z³represents X₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, orX₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined in claim 1; preferablyonly one of Z¹, Z² or Z³ represents X₁—R₃—X₂—P⁺, wherein X₁ representsCH₂, X₂ represents CO, and R₃ represents (CH₂)_(m), with m representingan integer between 1 and 3, preferably m is equal to 2; and/or (ii) Z¹represents H; and/or (iii) Z² represents H, a C₁-C₁₂ alkoxy, or a linearor branched, saturated or unsaturated C₁-C₁₈ alkyl, preferably a linearor branched, saturated or unsaturated C₁-C₆ alkyl; more preferably Z²represents H, CH₃, CF₃ or OCH₃; and/or (iv) Z³ represents H, or a linearor branched, saturated or unsaturated C₁-C₁₈ alkyl, preferably a linearor branched, saturated or unsaturated C₁-C₆ alkyl.
 6. The compositionaccording to claim 1, wherein: if (i) Z¹ represents X₁—R₃—X₂—P⁺,X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺, preferablyX₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein; morepreferably Z¹ represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂represents CO, and R₃ represents (CH₂)_(m), with m representing aninteger between 1 and 3, preferably m is equal to 2 then (ii) Z²represents H, a C₁-C₁₂ alkoxy, or a linear or branched, saturated orunsaturated C₁-C₁₈ alkyl, preferably a linear or branched, saturated orunsaturated C₁-C₆ alkyl; more preferably Z² represents H, CH₃, CF₃ orOCH₃; and/or (iii) Z³ represents H, a linear or branched, saturated orunsaturated C₁-C₁₈ alkyl, preferably a linear or branched, saturated orunsaturated C₁-C₆ alkyl, or a linear or branched, saturated orunsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, preferably fluorobenzyl or4-hydroxyphenethyl; and/or (iv) R or V represents H, a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl or cycloalkyl, a C₆-C₁₈aryl, a linear or branched, saturated or unsaturated C₆-C₁₈ aryl-C₁-C₁₈alkyl, a linear or branched, saturated or unsaturated C₂-C₁₈heteroalkyl, a linear or branched, saturated or unsaturated C₁-C₂₄ester, a C₅-C₁₀ heterocyclyl, a C₅-C₁₀ heteroaryl, or a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl-C₅-C₁₀ heteroaryl. 7.The composition according to claim 1, wherein: if (i) Z² representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein;more preferably Z² represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂represents CO, and R₃ represents (CH₂)_(m), with m representing aninteger between 1 and 3, preferably m is equal to 2 then (ii) Z¹represents H; and/or (iii) Z³ represents H, a linear or branched,saturated or unsaturated C₁-C₁₈ alkyl, preferably a linear or branched,saturated or unsaturated C₁-C₆ alkyl, or a linear or branched, saturatedor unsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, preferably fluorobenzyl or4-hydroxyphenethyl; and/or (iv) R or V represents H, a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl or cycloalkyl, a C₆-C₁₈aryl, a linear or branched, saturated or unsaturated C₆-C₁₈ aryl-C₁-C₁₈alkyl, a linear or branched, saturated or unsaturated C₂-C₁₈heteroalkyl, a linear or branched, saturated or unsaturated C₁-C₂₄ester, a C₅-C₁₀ heterocyclyl, a C₅-C₁₀ heteroaryl, or a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl-C₅-C₁₀ heteroaryl. 8.The composition according to claim 1, wherein: if (i) Z³ representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein;more preferably Z³ represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂represents CO, and R₃ represents (CH₂)_(m), with m representing aninteger between 1 and 3, preferably m is equal to 2 then (ii) Z¹represents H; and/or (iii) Z² represents H, a C₁-C₁₂ alkoxy, or a linearor branched, saturated or unsaturated C₁-C₁₈ alkyl, preferably a linearor branched, saturated or unsaturated C₁-C₆ alkyl; more preferably Z²represents H, CH₃, CF₃ or OCH₃; and/or (iv) R or V represents H, alinear or branched, saturated or unsaturated C₁-C₁₈ alkyl or cycloalkyl,a C₆-C₁₈ aryl, a linear or branched, saturated or unsaturated C₆-C₁₈aryl-C₁-C₁₈ alkyl, a linear or branched, saturated or unsaturated C₂-C₁₈heteroalkyl, a linear or branched, saturated or unsaturated C₁-C₂₄ester, a C₅-C₁₀ heterocyclyl, a C₅-C₁₀ heteroaryl, or a linear orbranched, saturated or unsaturated C₁-C₁₈ alkyl-C₅-C₁₀ heteroaryl. 9.The composition according to claim 1, wherein: if (i) R or V representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein;more preferably Z³ represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂represents CO, and R₃ represents (CH₂)_(m), with m representing aninteger between 1 and 3, preferably m is equal to 2 then (ii) Z¹represents H; and/or (iii) Z² represents H, a C₁-C₁₂ alkoxy, or a linearor branched, saturated or unsaturated C₁-C₁₈ alkyl, preferably a linearor branched, saturated or unsaturated C₁-C₆ alkyl; and/or (iv) Z³represents H, a linear or branched, saturated or unsaturated C₁-C₁₈alkyl, preferably a linear or branched, saturated or unsaturated C₁-C₆alkyl, or a linear or branched, saturated or unsaturated C₆-C₁₈aryl-C₁-C₁₈ alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl. 10.The composition according to claim 1, wherein: if (i) R or V representsX₁—R₃—X₂—P⁺, X₁—R₃—P⁺, X₁—X₂—P⁺, R₃—X₂—P⁺, X₁—P⁺, R₃—P⁺, or X₂—P⁺,preferably X₁—R₃—X₂—P⁺, wherein X₁, X₂, R₃ and P⁺ are as defined herein;more preferably Z³ represents X₁—R₃—X₂—P⁺, wherein X₁ represents CH₂, X₂represents CO, and R₃ represents (CH₂)_(m), with m representing aninteger between 1 and 3, preferably m is equal to 2 then (ii) Z³ ispresent and Z³ represents H, a linear or branched, saturated orunsaturated C₁-C₁₈ alkyl, preferably a linear or branched, saturated orunsaturated C₁-C₆ alkyl, or a linear or branched, saturated orunsaturated C₆-C₁₈ aryl-C₁-C₁₈ alkyl, preferably fluorobenzyl or4-hydroxyphenethyl.
 11. The composition according to claim 1, whereinthe graft cationic polymer is selected from the group consisting of alinear or branched polyethyleneimine (PEI), PEI dendrimers, apolypropyleneimine (PPI), Poly(amidoamine) (PAA) and dendrimers (PAMAM),cationic cyclodextrin, polyalkylamine, a polyhydroxyalkylamine,poly(butyleneimine) (PBI), spermine, a N-substituted polyallylamine,N-substituted chitosan, a N-substituted polyornithine, a N-substitutedpolylysine (PLL), a N-substituted polyvinylamine, poly(β-amino ester),hyperbranched poly(amino ester) (h-PAE), networked poly(amino ester)(n-PAE), poly(4-hydroxy-1-proline ester) (PHP-ester) and apoly-β-aminoacid.
 12. The composition according to claim 11, wherein thegraft cationic polymer is a linear or branched PEI, more preferably alinear PEI.
 13. The composition according to claim 1, wherein the graftcationic polymer has a grafting ratio ranging from 1 to 50%, preferablyfrom 5 to 30%, more preferably is 20%.
 14. The composition according toclaim 1, wherein the graft cationic polymer has an average molecularweight (Mw) ranging from 1 kDa to 500 kDa, preferably from 1 kDa to 50kDa, more preferably from 5 kDa to 50 kDa or from 1 kDa to 15 kDa. 15.The composition according to claim 14, wherein the graft cationicpolymer has an average molecular weight (Mw) of 6, 8, 10, 15, 22 or 30kDa, preferably of 6, 8, 10, 15 or 30 kDa.
 16. The composition accordingto claim 1, wherein the at least one compound of general formula (III)is selected from the group consisting of the following compounds:


17. The composition according to claim 16, wherein the at least onecompound of general formula (III) is selected from the group consistingof the following compounds:


18. The composition according to claim 17, wherein the at least onecompound of general formula (III) is compound 2.22.
 19. A method for invitro or ex vivo transfection of live cells comprising introducing inthe cells the composition according to claim
 2. 20. A method for invitro or ex vivo transfection of at least one nucleic acid molecule intoa cell, cell line or cells, preferably a cell, cell line or cellsselected from the group consisting of a mammalian cell, an insect cell,a primary cell, an adherent cell, a suspension cell, a dividing cellsuch as a stem cell, a non-dividing cell such as a neuronal cell, and acancer cell, said cell, cell line or cells being optionally organizedinto spheroids, organoids, 2D or 3D cell culture, or provided as fibreor matrix culture, and/or within a bioreactor, the method comprisingintroducing the composition of claim 2 into the cell, the cell line, orthe cells.
 21. A method for genome engineering, for cell reprogramming,for differentiating cells, or for gene-editing, comprising applying tothe genome, cells, or gene the composition according to claim
 2. 22. Amethod for the production of: (i) biologics, in particular biologicsencoding a recombinant protein, peptide or antibody, the methodcomprising applying the composition of claim 2; or (ii) recombinantvirus, such as adeno-associated virus (AAV), lentivirus (LV),adenovirus, oncolytic virus, or baculovirus, the method comprisingapplying the composition of claim 2, said composition comprisingmultiple nucleic acid molecules for co-transfection; or (iii) viral orvirus-like particles, the method comprising applying the compositionaccording to claim 2, said composition comprising multiple nucleic acidmolecules for co-transfection.
 23. The method according to claim 22, forthe production of AAV, said composition comprising (i) at least onecompound selected from the group consisting of compounds 2.22, 2.23,2.43, 2.44, 2.47, 2.54, 2.57, 2.60 and 2.61 and (ii) an acceptableexcipient, buffering agent, cell culture medium, or transfection medium.24. The method according to claim 22, for the production of LV, saidcomposition comprising (i) at least the compound 2.22, and (ii) anacceptable excipient, buffering agent, cell culture medium, ortransfection medium.
 25. The method according to claim 22, for theproduction of recombinant virus, said composition comprising a pluralityof expression vectors such as plasmid vectors to transfect in anadherent or suspension cell, such as HEK293, HeLa, BHK-21, A549 orinsect cells, wherein said vectors, in particular plasmids, areconstruct expressing viral structural sequences and transfer vectorgenome for virus or virus-like production and optionally expressingmolecules of interest encoded by the transfer vector genome.
 26. Themethod according to claim 25, further comprising a step of performingcell therapy or gene therapy, wherein the recombinant virus is used invivo.